Deliberate Self-poisoning with a Lethal Dose of Pentobarbital with Confirmatory Serum Drug Concentrations: Survival After Cardiac Arrest with Supportive Care.

INTRODUCTION: Pentobarbital (PB) is a euthanasia drug in doses of 2 to 10 grams, causing death within 15-30 minutes. We report a case of recovery from lethal pentobarbital deliberate self-poisoning with confirmatory serum drug concentrations. CASE REPORT: A 45-year-old male purchased 20 grams of PB powder over the Internet. He ingested this powder and then alerted his mother 10 minutes later. She found him unresponsive and commenced cardiopulmonary resuscitation (CPR). Within 20 minutes of ingestion, emergency medical services arrived and initiated advanced life support. On arrival to the emergency department, heart rate was 116 bpm, BP 117/62 mmHg, on an epinephrine infusion. He was hypotonic and hypothermic, with absent brainstem reflexes. ECG and CT brain were normal. Activated charcoal was administered and he was admitted to ICU. He remained comatose with absent brainstem reflexes until day 5. Cerebral angiogram on day 3 was normal. Qualitative urine testing detected pentobarbital suggesting ongoing drug effects as the cause of coma. He was extubated on day 10, eventually making a full recovery. At 2.5 hours post-ingestion, PB concentration was 112 mg/L; PB peaked at 116 mg/L at 29 hours; PB was 2 mg/L at 190 hours and undetectable over 200 hours post-ingestion. DISCUSSION: Average PB concentration in fatalities is reported around 30 mg/L. This patient survived higher serum concentrations with early CPR and prolonged cardiorespiratory support in the ICU. Assessment of brainstem death should be deferred until PB has been adequately eliminated.

Suicide by Fatal Pentobarbital Intoxication in Ontario, Canada, from 2012 to 2015.

A fatal concentration of pentobarbital found in a coroner's case where the history had not indicated use of this drug prompted a review of fatalities in Ontario from 2012 to 2015. Coroner's case files, including police and toxicology reports, were reviewed in twenty deaths, in which pentobarbital was identified as the primary cause of death. In all of the deaths (11 females, 9 males), the blood concentration of pentobarbital was greater than 10 mg/L. There were three to eight deaths per year and each was classified as suicide. In 11 cases, there was clear evidence that the drug was purchased over the internet from Mexico or China and imported into Canada. In four cases, it appears that the pentobarbital was labeled as a different, innocuous chemical to facilitate crossing the border without scrutiny. The findings underscore the value of a thorough scene investigation, including details of evidence that may be considered unrelated.

Interaction between different extracts of Hypericum perforatum L. from Serbia and pentobarbital, diazepam and paracetamol.

Herb-drug interactions are an important safety concern and this study was conducted regarding the interaction between the natural top-selling antidepressant remedy Hypericum perforatum (Hypericaceae) and conventional drugs. This study examined the influence of acute pretreatment with different extracts of Hypericum perforatum from Serbia on pentobarbital-induced sleeping time, impairment of motor coordination caused by diazepam and paracetamol pharmacokinetics in mice. Ethanolic extract, aqueous extract, infusion, tablet and capsule of Hypericum perforatum were used in this experiment. The profile of Hypericum perforatum extracts as well as paracetamol plasma concentration was determined using RP-HPLC analysis. By quantitative HPLC analysis of active principles, it has been proven that Hypericum perforatum ethanolic extract has the largest content of naphtodianthrones: hypericin (57.77 µg/mL) and pseudohypericin (155.38 µg/mL). Pretreatment with ethanolic extract of Hypericum perforatum potentiated the hypnotic effect of pentobarbital and impairment of motor coordination caused by diazepam to the greatest extent and also increased paracetamol plasma concentration in comparison to the control group. These results were in correlation with naphtodianthrone concentrations. The obtained results have shown a considerable influence of Hypericum perforatum on pentobarbital and diazepam pharmacodynamics and paracetamol pharmacokinetics.

Population pharmacokinetics of pentobarbital in neonates, infants, and children after open heart surgery.

OBJECTIVES: To determine the pharmacokinetics of pentobarbital in neonates, infants, and young children with congenital heart disease after open-heart surgery. STUDY DESIGN: Thirty-five subjects (3.0 days-4.4 years) after open-heart surgery who received pentobarbital as standard of care were enrolled. Serial pharmacokinetic blood samples were obtained. A population-based, nonlinear mixed-effects modeling approach was used to characterize pentobarbital pharmacokinetics. RESULTS: A two-compartment model with weight as a co-variate allometrically expressed on clearance (CL), inter-compartmental clearance, central (V1) and peripheral volume of distributions, bypass grafting time as a co-variate on CL and V1, and age and ventricular physiology as co-variates on CL best described the pharmacokinetics. A typical infant (two-ventricle physiology, 6.9 kg, 5.2 months, and bypass grafting time of 60 minutes) had a CL of 0.12 L/hr/kg, V1 of 0.45 L/kg, and peripheral volume of distributions of 0.98 L/kg. The bypass grafting effect was poorly estimated. For subjects <12 months age, an age effect on CL remained after accounting for weight and was precisely estimated. CONCLUSIONS: Pentobarbital pharmacokinetics is influenced by age and weight. Subjects with single-ventricle physiology demonstrated a 15% decrease in clearance when compared with subjects with two-ventricle physiology.

Secondary pentobarbital poisoning in two dogs: a cautionary tale.

Two dogs, a 13-year-old spayed female and a 7-year-old neutered male, were diagnosed with pentobarbital poisoning. Follow-up investigation determined that the source of pentobarbital was the carcass of a horse that had been euthanized more than 2 years previously and that was also apparently responsible for the death of a least 1, and possibly 2, other dogs. The fact that the horse carcass remained lethally toxic more than 2 years after it was euthanized reemphasizes the necessity of proper disposal of euthanized animals.

Quantitation of amobarbital, butalbital, pentobarbital, phenobarbital, and secobarbital in urine, serum, and plasma using gas chromatography-mass spectrometry (GC-MS).

Barbiturates are central nervous system depressants with sedative and hypnotic properties. Some barbiturates, with longer half-lives, are used as anticonvulsants. Their mechanism of action includes activation of gamma-aminobutyric acid (GABA) mediated neuronal transmission inhibition. Clinically used barbiturates include amobarbital, butalbital, pentobarbital, phenobarbital, secobarbital, and thiopental. Besides their therapeutic use, barbiturates are commonly abused. Their analysis is useful for both clinical and forensic proposes. Gas chromatography mass spectrometry is a commonly used method for the analysis of barbiturates. In the method described here, barbiturates from serum, plasma, or urine are extracted using an acidic phosphate buffer and methylene chloride. Barbital is used as an internal standard. The organic extract is dried and reconstituted with mixture of trimethylanilinium hydroxide (TMAH) and ethylacetate. The extract is injected into a gas chromatogram mass spectrometer where it undergoes "flash methylation" in the hot injection port. Selective ion monitoring and relative retention times are used for the identification and quantitation of barbiturates.

Head injuries, pentobarbital, and the determination of death.

The Bexar County Medical Examiner's Office in San Antonio, Texas, has encountered 3 cases within a 15-month period involving decedents who were pronounced dead by brain death or cardiac death examination and who had elevated, if not toxic concentrations of pentobarbital present at the time of examination. The elevated levels of pentobarbital were discovered during an autopsy examination performed for medicolegal reasons. The diagnosis of brain death and the implications of pentobarbital intoxication during a brain death examination are discussed.

Drugs and brain death diagnostics: determination of drugs capable of inducing EEG zero line.

BACKGROUND: Several drugs may affect the diagnosis of brain death by depressing the electroencephalographic signal. Serum levels of these drugs must be below their respective therapeutic ranges. METHODS: A high performance liquid chromatography-based fast and simple method was developed for determination of thiopentone, pentobarbitone, phenobarbitone, methohexital and propofol in serum and validated according to international recommendations. RESULTS: Separation of extracted analytes was performed on a reversed phase column [Agilent Zorbax SB C18, 5 microm, 4.6 x 250 mm; mobile phase 50% 50 mM NaH(2)PO(4) pH 4.6 mixed with 35% (v/v) acetonitrile and 15% (v/v) methanol]. Calibration curves were linear throughout the selected ranges (microg/mL, thiopentone 0.25-50, pentobarbitone 0.25-25, phenobarbitone 2.5-50, methohexital 0.125-2.50, propofol 0.25-5.0). The standard deviations for the regression line, recovery, imprecision and accuracy results were all highly satisfactory. The lower limits of quantification for propofol, thiopentone and pentobarbitone were set at 0.25 microg/mL, for phenobarbitone 2.5 microg/mL, and for methohexital 0.125 microg/mL, which are below the lowest pharmacologically relevant serum concentrations. Intra- and inter-day coefficients of variation were less than 10% throughout as determined with six replicates. CONCLUSIONS: The method presented is suitable for drug monitoring to help enhance the reliability of the diagnosis of brain death.

Usefulness of serum concentration measurement for acute pentobarbital intoxication in patients.

Toxicokinetic parameters were analyzed in 25 patients who were acutely intoxicated with pentobarbital. The serum pentobarbital concentrations were at hypnotic / therapeutic levels (1- 10 microg/mL) in 11 patients, at toxic levels (10-24 microg/mL) in 10 patients, and at lethal levels (>24 microg/mL) in 4 patients. One fatal case was encountered with a serum pentobarbital concentration of 37.27 microg/ mL. The relationships between serum pentobarbital concentrations and the drug, the induced toxic symptoms, such as respiratory / cardiac depression and hypotension, were in good correlation. The therapy of direct hemoperfusion carried out for many pentobarbital-intoxicated patients was highly effective.

More reliable brain death diagnosis with chromatographic analysis of midazolam, diazepam, thiopentone, and active metabolites.

Brain death diagnosis may be confounded by centrally acting drugs. The certainty of brain death diagnosis can be enhanced by demonstrating that the concentrations of such drugs are well below the therapeutic range. A combined high-performance liquid chromatography-based method was developed for the benzodiazepines midazolam, 1-hydroxymidazolam, 1-hydroxymidazolam glucuronide, diazepam, and nordiazepam and for the barbiturates thiopentone and pentobarbitone in serum or plasma of critically ill patients. The lower limits of detection of the assays for benzodiazepines and barbiturates were 2.5 ng/mL and 0.05 microg/mL. The lower limits of the working ranges of these assays were set at 25 ng/mL and 0.5 microg/mL, respectively, and are below the lowest pharmacologically active plasma concentrations of these drugs. Intra- and interday coefficients of variations were less than 2.5% and 11.0% throughout, as determined with six replicates (n = 6). These assays were accurate in that the relative difference between actually measured and expected concentration never exceeded 12%. Utilization of these assays will render the diagnosis of brain death more reliable.

Method for screening and quantitative determination of serum levels of salicylic Acid, acetaminophen, theophylline, phenobarbital, bromvalerylurea, pentobarbital, and amobarbital using liquid chromatography/electrospray mass spectrometry.

We investigated a method for the simultaneous screening, identification, and quantitative determination of salicylic acid, acetaminophen, theophylline, barbiturates, and bromvalerylurea, drugs that frequently cause acute poisoning in Japan and therefore require rapid analysis for effective treatment in the clinical setting. The method employs liquid chromatography/electrospray mass spectrometry (LC/MS) of solid-phase extracted serum samples. For LC/MS ionization, the electrospray-ionization method was used, with acetaminophen in the positive-ion mode, and salicylic acid, theophylline, phenobarbital, bromvalerylurea, pentobarbital, amobarbital, and o-acetamidophenol (internal standard) in the negative-ion mode, the base ions were used in each case for quantitative analysis. Quantitation was possible for the following sample concentration ranges: salicylic acid and acetaminophen, 100 to 5 microg/ml; theophylline, 100 to 0.5 microg/ml; and phenobarbital, bromvalerylurea, pentobarbital, and amobarbital, 100 to 1 microg/ml. Using full-scan mass spectrometry, the lower detection limits of 1 microg/ml for salicylic acid and acetaminophen, 0.1 microg/ml for theophylline, and 0.5 microg/ml for phenobarbital, bromvalerylurea, pentobarbital, and amobarbital were adequate for identifying acute poisoning. When each compound was added to serum to a final concentration of 5 microg/ml and solid-phase extraction was performed using Oasis HLB 1-cc (30-mg), the mean recovery rate of each compound was 89.2 to 96.1% (n=5), and the coefficients of variation of the intraday and interday assays were 3.55 to 6.05% (n=5) and 3.68 to 6.38% (n=5), respectively, which are acceptable. When this method of analysis was applied in testing the sera of a female patient who had consumed a large amount of an unknown commercial drug, salicylic acid and bromvalerylurea were identified, and the treatment strategy could be determined in accordance with the serum concentration of those drugs.

Comparing the bispectral index and suppression ratio with burst suppression of the electroencephalogram during pentobarbital infusions in adult intensive care patients.

OBJECTIVE: The bispectral index (BIS), a processed variable derived from the raw electroencephalogram (EEG) used to guide sedation in the intensive care unit (ICU), has not been tested during barbiturate therapy for elevated intracranial pressure. We determined the BIS and suppression ratio (SR) values during traditional burst monitoring of the raw EEG during pentobarbital infusions. DESIGN: Prospective, observational cohort study. SETTING: A 42-bed multidisciplinary ICU in a tertiary care medical center. PATIENTS: Twelve consecutive patients with elevated intracranial pressure treated with pentobarbital infusions. INTERVENTION: All patients were monitored continuously with the Aspect Medical Systems A-1050 bedside EEG monitor using a bilateral referential montage. Pentobarbital doses were titrated based on the raw EEG to attain a burst-suppression pattern with a goal of 3-5 bursts/minute. Drug dosage, intracranial pressure, cerebral perfusion pressure values, EEG bursts/minute, BIS version 3.2, and SR were recorded daily. MEASUREMENTS AND MAIN RESULTS: The 12 patients were monitored for 62 patient-days. Mean +/- SD age was 32 +/- 15 years, seven (58%) patients were male, mean Acute Physiology and Chronic Heath Evaluation II score was 17.0 +/- 5.0, and hospital mortality was 42%. The mean pentobarbital infusion rate was 124 +/- 49 mg/hour or 2.3 +/- 1.3 mg/kg/hour, and mean pentobarbital serum concentration was 29.7 +/- 13 microg/ml. The mean BIS value was 18 +/- 14, mean SR 56% +/- 36%; BIS correlated well with SR (r=-0.99, p<0.001). For patient-days with a burst-suppression pattern, BIS 3.2 (r=0.90, p<0.001) and SR (r=-0.89, p<0.001) strongly correlated with the number of bursts/minute. The mean BIS value corresponding to 3-5 bursts/minute was 15 (95% confidence interval [CI] 10-20); SR value was 71 (95% CI 61-80). CONCLUSION: The Aspect A-1050 applied to patients and monitored by nurses and physicians works well as a bedside EEG monitor, providing a raw EEG signal to titrate barbiturate therapy. The continuous data trend and real-time digital output for the BIS and SR quantify the degree of EEG suppression well and may prove helpful in facilitating titration of barbiturate infusions.

Blockade of glutamate receptors and barbiturate anesthesia: increased sensitivity to pentobarbital-induced anesthesia despite reduced inhibition of AMPA receptors in GluR2 null mutant mice.

BACKGROUND: Barbiturates enhance gamma-aminobutyric acid type A (GABA(A)) receptor function and also inhibit the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates. METHODS: GluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABA(A) receptors. RESULTS: Behavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 microM), thiopental (IC50, 153 vs. 34 microM), and phenobarbital (IC50, 930 vs. 205 microM) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABA(A) receptors by pentobarbital. CONCLUSIONS: The GluR2 null mutant mice were more sensitive to pentobarbital anesthesia despite a reduced sensitivity of GluR2-deficient AMPA receptors to barbiturate blockade. Our results indicate that the inhibition of AMPA receptors does not correlate with the anesthetic effects of barbiturates in this animal model. We postulate that the increase in the sensitivity to anesthetics results from a global suppression of excitatory neurotransmission in GluR2-deficient mice.

Comparison of the serum barbiturate fluorescence polarization immunoassay by the COBAS INTEGRA to a GC/MS method.

The authors evaluated a new Cassette Serum Barbiturates fluorescence polarization immunoassay (FPIA) on the COBAS INTEGRA. The assay was calibrated with secobarbital standards at 0, 0.5, and 4.0 microg/mL. The assay range was 0.030 to 80 microg/mL using an automatic 1/20 postdilution feature. Precision was established for two COBAS INTEGRA instruments for ten days by assaying secobarbital target concentrations ranging from 0.125 to 2.2 microg/mL. The coefficients of variation (CV) for the above target concentrations for the first instrument ranged from 2.7% to 8.3%, and for a second instrument, 3.8 to 8.3%. Seven clinically elevated bilirubin samples were spiked with 0.46 and 1.77 microg/mL secobarbital. Bilirubin interference was less than 10.9 and less than 7.9%, respectively. The average recovery ranged from 85% to 94%. The mean difference in recovery in serum versus plasma was < or = 3%. Fifty-two clinical samples were analyzed for butalbital, pentobarbital, secobarbital, and phenobarbital by GC/MS, and the results were compared to the new Cassette Serum Barbiturates FPIA. The diagnostic sensitivity and specificity were 95% and 100%, respectively. Both FPIA and GC/MS assays are clinically efficacious for monitoring serum barbiturates.

Stability of thiopental and pentobarbital in human plasma determined with a new easy and specific gas chromatography-mass spectrometry assay.

A gas chromatographic-mass spectrometric (GC-MS) assay for the determination of thiopental and its main metabolite pentobarbital in human plasma is presented in this study. The sample preparation consists only in the addition of the internal standard barbital and an acidic extraction with ethyl acetate. Analytical separation is accomplished on a RTX-1 15 m x 0.25 mm capillary column with a film thickness of 0.5 micron. The effluent is observed by a mass selective detector operating in the single ion monitoring mode. The limits of detection are 5 ng/ml for pentobarbital and 10 ng/ml for thiopental, the intra-day variabilities are 2.2% and 4.0% and the inter-day variabilities are 3.3% and 7.1% at concentrations of 5 micrograms/ml, respectively. Applying this assay, the stability of thiopental and pentobarbital in human plasma was tested at concentrations of 5 micrograms/ml each. Thiopental is stable in human plasma at least over 41 days stored at -20 degrees C and 5 degrees C, respectively. A decay of about 2%/day is observed under storage at ambient temperature (19-20 degrees C). Pentobarbital is stable under all storage conditions. Methanolic solutions of thiopental are stable for 83 days under storage at 5 degrees C. Aqueous solutions of thiopental-sodium are stable for at least 23 days under storage at 5 degrees C or ambient temperature.

Characterization of the stereoselective metabolism of thiopental and its metabolite pentobarbital via analysis of their enantiomers in human plasma by capillary electrophoresis.

Using capillary zone electrophoresis (CZE) with a 75 mM phosphate buffer at pH 8.5 containing 5 mM hydroxypropyl-gamma-cyclodextrin (OHP-gamma-CD) as chiral selector, the separation of the enantiomers of thiopental and its oxybarbiturate metabolite, pentobarbital, is reported. Enantiomer assignment was performed via preparation of enantiomerically enriched fractions using chiral recycling isotachophoresis (rITP) processing of racemic barbiturates and analysis of rITP fractions by chiral CZE and circular dichroism spectroscopy. Thiopental and pentobarbital enantiomers in plasma were extracted at low pH using dichloromethane and extracts were reconstituted in acetonitrile or 10-fold diluted, achiral running buffer. The stereoselectivity of the thiopental and pentobarbital metabolism was assessed via analysis of 12 plasma samples that stemmed from patients undergoing prolonged or having completed long-term racemic thiopental infusion. The data obtained revealed a modest stereoselectivity with R-(+)-thiopental/S-(-)-thiopental and R-(+)-pentobarbital/S-(-)-pentobarbital plasma ratios being < 1 (P < 0.05 compared to data obtained with racemic controls) and > 1 (P < 0.001), respectively. The total S-(-)-thiopental plasma concentration was found to be on average about 24% higher compared to the concentration of R-(+)-thiopental, whereas the total R-(+)-pentobarbital plasma level was observed to be on average 29% higher compared to the S-(-)-pentobarbital concentration.

Exit association-mediated suicide: toxicologic and forensic aspects.

The Swiss German chapter of the Exit Association provides conditional assistance to individuals wishing to end their own lives. The Exit Association advocates death with dignity and fights for the right to freely choose the timing of one's own death. According to the Swiss criminal code (articles 114 and 115), altruistic assistance to suicide is not punishable. Active euthanasia is punished by imprisonment. An individual commits active euthanasia if he or she is driven by honorable motives (e.g., pity) and causes the death of another person wishing to die who seriously and insistently requests such action. Based on our information, the preparation for suicide and its completion relies on a well-defined protocol. First, the candidate's eligibility for Exit Association assistance is verified. The candidate then writes a farewell declaration that explicitly confirms the will to terminate his or her own life. A written report describes the events during the suicide procedure. Depending on the circumstances, the investigative judge requests a forensic autopsy and toxicologic analyses. The results of the forensic investigations conducted in the cases presented here are in agreement with the scenario described in the reports of the Exit Association, namely, suicide by massive ingestion of pentobarbital.

Effects of muscle relaxants on EEG, ABR and EMG in rabbits.

The effects of baclofen and pancuronium bromide on evoked electromyogram (EMG), cortical electroencephalogram (EEG) and auditory brainstem responses (ABR) were studied in pentobarbital anesthetized normal rabbits. Evoked EMG was measured in the gastrocnemius muscle by electrical stimulation of the sciatic nerve. Intravenous injection of baclofen decreased EEG and arterial blood pressure and light reflex, however, it had no significant influence on EMG or ABR at doses of 10 and 20 mg/kg/h. Pancuronium bromide immediately inhibited respiration, decreased EEG and EMG, however, it had no significant influence on arterial blood pressure, ABR, or light reflex, at doses of 0.4 and 1.0 mg/kg/h in anesthetized rabbits. ABR waves were observed until just before cardiac arrest with both of the muscle relaxants. It is suggested that ABR are not influenced by central or peripheral muscle relaxants, or by pentobarbital.

Supercritical fluid extraction and negative ion electrospray liquid chromatography tandem mass spectrometry analysis of phenobarbital, butalbital, pentobarbital and thiopental in human serum.

Four commonly used barbiturates (phenobarbital, butalbital, pentobarbital and thiopental) were analyzed in human serum using supercritical fluid extraction (SFE) and negative ionization LC/ESI-MS/MS. Barbital was used as the internal standard. Carbon dioxide SFE was performed at 40 degrees C and 500 atm, with a total extraction time of 35 min. The analytes were collected off-line in a liquid trap containing absolute methanol. Samples were then concentrated by vacuum centrifugation. The high performance liquid chromatography separation utilized gradient elution with a total analysis time of 21 min. The precursor and major product ions for the four barbiturates were monitored on a triple quadrupole mass spectrometer with negative ion electrospray ionization (ESI) in the multiple reaction monitoring mode as follows: (1) thiopental (m/z 241.20-->58.00), (2) phenobarbital (m/z 231.10-->188.0), (3) pentobarbital (m/z 225.10-->181.90) and (4) butalbital (m/z 222.80-->179.90). In the case of phenobarbital, pentobarbital and butalbital, the most abundant product ion arises from the loss of 43 u (HCNO loss). However, in the case of thiopental, the most abundant product ion was observed at m/z 58.0 (the [M-183]-ion, or NCS-). Mechanisms for the formation of the collision induced dissociation reaction products of these barbiturates are proposed.