Disposition of methadone in the ovine maternal-fetal unit

The distribution of methadone between mother and fetus after a single dose and at steady state was determined using the chronic pregnant ewe preparation. Chronic indwelling catheters were placed in the maternal aorta and vena cava, umbilical vein and fetal aorta. Following a single i.v. dose (0.5 mg/kg) to the mother, methadone was rapidly distributed to the fetus, with peak concentration in the umbilical vein occurring within two min. An umbilical venous-arterial gradient existed for 10–15 min after drug administration, indicating uptake of methadone by fetal tissues. Methadone concentration in the fetus was 2–5 times lower than those in the mother even in the post-distribution phase. The terminal half-life of methadone in 4 animals was 57±7.6 (S.E.) min in the mother, and 58.5±10.0 (S.E.) min in the fetus. When methadone was infused at a constant rate to the mother (0.01 mg/kg/min), steady state was achieved in both mother and fetus by 4–5 hrs. In 5 animals, maternal steady state was found to be 203±18.8 (S.E.) ng/ml, and fetal steady state was found to be 29.7±2.9 (S.E.) ng/ml. These studies show that methadone is rapidly distributed to the fetus, but fetal concentration remain lower than maternal concentration at all times.     

Method for determination of methadone in exhaled breath collected from subjects undergoing methadone maintenance treatment

At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use.     

Adipocyte-derived hormones in heroin addicts: the influence of methadone maintenance treatment

Heroin addiction markedly affects the nutritional and metabolic status and frequently leads to malnutrition. The aim of our study was to compare circulating concentration of adipose tissue-derived hormones leptin, adiponectin and resistin in 12 patients with heroin addiction before and after one-year methadone maintenance treatment with the group of 20 age- and body mass index-matched healthy subjects. Basal serum leptin and adiponectin levels in heroin addicts were significantly decreased (3.4+/-0.4 vs. 4.5+/-0.6 ng/ml and 18.9+/-3.3 vs. 33.9+/-3.1 ng/microl, respectively; p 0.05) while serum resistin concentrations were increased compared to healthy subjects (10.1+/-1.2 vs. 4.6+/-0.3 ng/ml; p 0.05). Moreover, positive correlation of serum leptin levels with body mass index was lost in the addicts in contrast to control group. One year of methadone maintenance treatment normalized serum leptin, but not serum adiponectin and resistin concentrations. In conclusion, circulating concentrations of leptin, adiponectin and resistin are markedly altered in patients with chronic heroin addiction. These alterations appear to be relatively independent of nutritional status and insulin sensitivity.     

Determination of methadone and its metabolites EDDP and EMDP in human hair by headspace solid-phase microextraction and gas chromatography–mass spectrometry

A simple method for analysis of methadone and its two main metabolites EDDP and EMDP in hair was developed using automatic headspace solid-phase microextraction (HS-SPME) at a multipurpose sampler and gas chromatography – mass spectrometry with electron impact ionization and selected ion monitoring (GC–MS-SIM). The washed hair pieces were digested in the closed headspace vial in 1 ml 1 M NaOH containing 0.5 g NaCl and each 10 ng of the internal standards D₉-methadone and D₃-EDDP at 110°C for 20 min. Then the HS-SPME was performed with a 65 μm polydimethylsiloxan/divinylbenzene fiber at the same temperature in the same vial for another 20 min followed by the desorption in the GC injection port. The calibration curves were linear between 0.1 and 3 ng/mg (methadone and EMDP) and 10 ng/mg (EDDP) respectively, at higher concentrations a negative deviation from linearity was found. The detection limits were 0.03 ng/mg (methadone) and 0.05 ng/mg (EDDP and EMDP), and the reproducibility was 9.2% for methadone and 11.2% for EDDP (n=12). The method was applied to hair samples of 26 drug fatalities. 19 cases were positive with 0.36–11.8 ng/mg methadone and 0.19 –10.8 ng/mg EDDP. EMDP was found only in two cases with 0.18 and 0.84 ng/mg. The methadone concentration range was in agreement with previous data, but the EDDP/methadone concentration ratios (0.19–0.67) were definitely higher than those determined by other methods.     

Toxicology and pathology of deaths related to methadone: retrospective review

Objectives To clarify the mechanisms and risk factors of methadone toxicity and to describe the findings of deaths related to methadone use Design Retrospective review of case notes in the records of the San Francisco Medical Examiner comparing the findings in cases where methadone was deemed the cause of death with findings in decedents where methadone was an incidental finding, and with 50 age-matched, disease and drug free, trauma victims. Results 38 cases out of the 3317 processed by our office during 1997-1998 were identified in which methadone had been detected. Cases were mostly male 28/38 (74%) and white, 28/38 (74%). In 17 of 38 cases death was deemed to have been caused by methadone toxicity. For the group the mean blood methadone concentration for all 38 patients, was 957 ng/ml SD =.681, SE =.14). The mean blood concentration of the main methadone metabolite (EDDP) was 253 ng/ml, SD = 529 ng/ml, SE =.089. The mean ratio of methadone in the blood to EDDP in the blood was 13.6:1 Values were not significantly different between cases in which methadone toxicity was the cause of death and in those in which it was an incidental finding. Cocaine, or the cocaine metabolite benzoylecgonine, was detected in the blood or urine of 16/38 cases (42%); morphine in one-third (13/38) and methamphetamine in only one. Pulmonary edema was evident in all cases, coronary artery disease in 9/38 (24%) and cirrhosis in 7/38 (18%) of the methadone users. Necrotizing fasciitis was the cause of death in 4 of the 38 methadone users (11%). Nationally, a sizeable percent of methadone deaths are from drugs diverted from treatment programs. Conclusions The presence of methadone is often an incidental finding during postmortem examination which is unrelated to the cause of death. Postmortem measurements of methadone or its metabolite, or both, cannot be used in isolation to identify which deaths are associated with methadone toxicity.The Office on National Drug Control Policy is commited to making methadone treatment programs more widely available; deaths related to heroin use fall when where methadone replacement programs are available.^{1, 2, 3} Unfortunately, methadone is toxic. A total of 552 methadone-related deaths were reported to the government in 1996, making methadone the seventh most frequent cause of drug-related death in the United States (nearly 4000 deaths related to heroin were reported during that same period).⁴Most deaths that are related to methadone occur during the first few weeks of maintenance treatment; they are often the result of the dosage having been increased so quickly that fatal respiratory depression occurs.^5,6 The relative risk of fatal respiratory depression occurring during the first 2 weeks of methadone maintenance treatment is nearly seven times higher than that in untreated heroin addicts and 97.8 times higher than for patients who have been on methadone maintenance for more than 2 weeks.^3,7New opiate users who are using illicitly obtained methadone are also at risk. The amount of methadone diverted from treatment programs, and by inference the number of deaths occurring as a result, is limited because the number of heroin users actually enrolled in methadone programs is comparatively small. There are an estimated 810,000 heroin addicts in the United States but only 115,000 participate in maintenance programs.⁴ If methadone becomes more widely available opportunities for diversion from treatment programs will increase and so will the number of deaths.¹ Some of these deaths might be prevented, especially if the underlying cause of death was better understood. Little is known about the pharmacokinetics of methadone in opiate users. The data that have been published are largely derived from studies of single doses given to healthy volunteers or intravenous doses given to patients with cancer. Whether such studies are relevant to the pharmacokinetics in chronic heroin users is not known. Furthermore, nearly all of these studies were undertaken before differences in the tissue distribution of methadone isomers were understood,⁸ before methadone metabolites could be routinely measured, before chiral (special chemical techniques used to separate dextro- from levo- isomers of the same molecule) separation of methadone isomers was possible ⁹ and before the problem of determining the redistribution of drugs after death was appreciated.¹⁰     

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity     

Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19

Methadone is a clinically used opioid agonist that is oxidatively metabolized by cytochrome P450 (CYP) isoforms to a stable metabolite, EDDP. Methadone is a chiral drug administered as the racemic mixture of (R)-(-)- and (S)-(+)-methadone, but (R)-methadone is the active isomer. The cytochrome P450 (CYP) isoform involved in methadone's metabolism is thought to be CYP3A4, but human drug-drug interaction studies are not consistent with this. The ability of the common human drug-metabolizing CYPs (obtained from baculovirus-infected insect cell supersomes) to generate 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrilidine (EDDP) from racemic methadone was examined and then determined if the CYP isoforms metabolized methadone stereoselectively. Only CYP2B6, 2C19, and 3A4 generated measurable EDDP from 1 microg/ml of racemic methadone. The hierarchy of EDDP generation was CYP2B6 > CYP2C19 >/= CYP3A4. At 10 microg/ml of methadone, CYP2C9 and CYP2D6 also generated EDDP, but in at least 10-fold lower quantities than CYP2B6. Michaelis-Menten kinetic data demonstrated that CYP2B6 had the highest V(max) (44 ng/min/10pmol) and the lowest K(m) (12.6 microg/ml) for EDDP formation of all the CYP isoforms. In human liver microsomes with high and low CYP2B6 expression but equivalent CYP3A4 expression, high CYP2B6 expression microsomes generated twice the amount of EDDP from 10 microg/ml of methadone than low CYP2B6 expression microsomes. When stereoselective metabolism of racemic methadone by CYP2B6, 2C19, and 3A4 was examined using an enantiospecific methadone assay, CYP2B6 preferentially metabolized (S)-methadone, CYP2C19 preferentially metabolized (R)-methadone, and CYP3A4 showed no preference. These data suggest that multiple CYPs metabolized methadone but CYP2B6 had the highest V(max)/K(m). In addition, only CYP2B6 and 2C19 showed stereoselective metabolism. Our data could explain why the plasma concentration ratio of R/S methadone is variable and why drugs that induce CYP2B6 such as nevirapine and efavirenz also induce methadone metabolism, while the CYP3A4 inducer rifabutin has no effect on methadone pharmacokinetics.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane—isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound α₁-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.     

Determination of captopril in human plasma, using solid phase extraction and high-performance liquid chromatography, coupled to mass spectrometry: application to bioequivalence study

A specific high performance liquid chromatography-mass spectrometric (LC-MS/MS) assay was developed for the determination of captopryl in plasma. The retention time was 1.45 and 1.37 min for captopril and enalapril, respectively. The overall mean recovery, using SPE extraction with OASIS HLB cartridges, was found to be 107.2+/-9.5 and 100.04+/-2%, respectively. Calibration curves were linear in the concentration range of 10.00-2000.00 ng/ml, and the lower limit of quantification (LLOQ) was 10.00 ng/ml. The LLOQ was sensitive enough for detecting terminal phase concentrations of the drug. Inter-batch precision of the method ranged from 0.88 to 1.95%. Intra-batch accuracy ranged from 97.15 to 105.77%, while intra-batch precision ranged from 2.49 to 5.66% at concentrations of 30.00, 760.00 and 1500.00 ng/ml. The developed method was applied to study bioequivalence of captopril in a group of 25 human subjects at a single oral dose of a 50mg tablet.     

Follicle stimulating hormone and luteinizing hormone levels in buffalo seminal plasma

The levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were determined in the buffalo bull seminal plasma by double-antibody radioimmunoassay. The mean levels of FSH and LH ranged from 8.98 ± 3.08 to 18.40 ± 2.19 ng/ml and from 0.598 ± 0.200 to 1.22 ± 0.334 ng/ml, respectively. FSH and LH concentration was positively correlated with mass motility and sperm concentration of buffalo semen samples. Concentration of hormones did not differ significantly among bulls.     

Chiral analysis of methadone in plasma by high-performance liquid chromatography

A method for the chiral high-performance liquid chromatographic analysis of methadone in plasma has been developed. The method employed organic solvent extraction, enantiomeric separation on a Chiral AGP column, and ultraviolet absorption detection at 212 nm. The intra-day variation in the quantification of methadone enantiomers was less than 9% at the 100 ng/ml level, and the values obtained correlated well with those from a gas chromatographic—mass spectrometric method. Results from patients indicate inter- and intra-individual differences in the ratio between l- and d-methadone in plasma during therapy with racemic methadone. In one patient, a higher level of d-methadone in plasma was caused by both faster elimination and lower bioavailability of l-methadone.     

Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, 2-ethyl-5-methyl-3,3-diphenylpyraline and methadol in meconium by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

This paper details a validated liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) method for the quantification of methadone, and its metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenylpyraline (EMDP) and methadol in human meconium. Limits of detection (LOD) were determined to be 1.0 ng/g for methadone, EDDP and EMDP and 2.5 ng/g for methadol. The limits of quantitation (LOQ) for methadone, EDDP, EMDP were 5 and 25 ng/g for methadol. Linearity ranged from 5.0 to 500 ng/g. Following solid-phase extraction, no matrix effect was observed. This method proved to be suitable for the quantification of methadone, EDDP and EMDP and the semi-quantitation of methadol in meconium. Literature review revealed no other published LC-APCI-MS/MS method for the detection of methadone and its three main metabolites in meconium specimens.     

Immunoenzymatic method for determining thyroglobulin levels in human blood serum]

An inexpensive enzyme immunoassay method was designed for the determination of thyroglobulin concentration in human blood serum. The range of concentrations of thyroglobulin which can be measured by the method is between 6 and 800 ng/ml. The sensitivity of the method is comparable to that of the commercial test kits. The values of thyroglobulin concentration obtained with the use of the described method are strongly correlated (r = 0.946) with those obtained by using the reference method (IRMA kit of Byk, Sweden). The intraassay coefficient of variation ranged from 5.5 to 10.2% and interassay coefficient of variation from 9.5 to 13.2% depending on the thyroglobulin concentration. The upper limit of blood serum thyroglobulin concentration in healthy subjects was 70 ng/ml. The results of thyroglobulin determination obtained with the described method are falsely lowered in the presence of antithyroglobulin antibodies; simultaneous determination of these antibodies is thus necessary in such a case. It seems that the described method may be used for monitoring the patients after surgical treatment of differentiated thyroid cancer aimed at early detection of metastases.     

Freezability and Fertility Results with Uncentrifuged Stallion Semen

The first (1 to 3) sperm-rich fractions of the ejaculate were collected from 4 stallions using an open-ended vagina. The volume of the collected fractions was 12 ± 8 ml with a density of 475 ± 200 million spermatozoa/ml. Before freezing, the semen was diluted with a skim-milk based extender 1:1 to 1: 8 (volume of semen: volume of extender), depending on the initial sperm concentration to achieve a final concentration of 100 million/ml. The total number of spermatozoa in an insemination dose ranged from 0.7 to 1 billion spermatozoa. Within 12 h after ovulation, 48 mares were inseminated in 70 cycles. The total single-cycle pregnancy rate at day 21 was 24%, but varied from 10% to 33% per cycle among the stallions.     

Enhanced development of dispositional tolerance to methadone by desipramine given together with methadone

Rats given 2-day oral administration of methadone (15 mg/kg, twice on day 1 and once on day 2) by gastric tube developed dispositional tolerance to methadone analgesia as demonstrated by a decrease in analgesic response and by an increase in methadone metabolism. The increased metabolism of methadone was evidenced by a decrease in brain concentration of 14C-methadone and increases in the percentages of total 14C in liver or urine as 14C-water-soluble metabolites (14C-WSM) after the rats were challenged with a test dose of 14C-methadone. Two-day pretreatment with a combination of desipramine (DMI) (10 mg/kg, ip) and methadone (15 mg/kg, po) enhanced the development of dispositional tolerance to methadone analgesia which was evidenced by a greater decrease in the brain concentration of methadone and a greater increase in methadone metabolism as compared to those changes in rats pretreated with only methadone. Repeated treatment with DMI alone neither decreased the analgesic effect of methadone nor stimulated methadone metabolism. It is suggested that DMI given together with methadone promoted the induction of methadone metabolism in the liver by prolonging the enzyme-stimulating state of methadone, thus enhancing the development of dispositional tolerance to methadone.     

Gas chromatographic-mass spectrometric determination of ß2-agonists in postmortem blood: application in forensic medicine

A solid-phase extraction procedure is described for the simultaneous determination of terbutaline, salbutamol and fenoterol in human postmortem whole blood, using gas chromatography-electron impact mass spectrometry. The limit of quantitation in 1 ml of blood was 1 ng/ml for all analytes. A linear response was observed over the concentration ranges tested, covering both low and high concentration of each drug. The recoveries in postmortem blood were: terbutaline, 88%; salbutamol, 86%; fenoterol, 92%; orciprenaline (internal standard), 86%. Coefficients of variation for both intra-assay precision and inter-assay reproducibility ranged between 2.2 and 13.0% for all analytes. This method is sensitive and selective, and has been applied successfully to over 60 postmortem blood specimens.     

Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes

Methadone and buprenorphine are two of the drugs most frequently used for abstinence from illicit opioids and in the treatment of pain. A sensitive and selective high-performance liquid chromatographic method with diode array detection for the simultaneous determination of methadone, buprenorphine and norbuprenorphine has been developed. Separation of the three analytes was obtained by using a reversed-phase column (C8, 250mmx4.6mm i.d., 5microm) and a mobile phase composed of 40% phosphate buffer containing triethylamine, 50% methanol and 10% acetonitrile (final apparent pH 6.0). Loxapine was used as the internal standard. An accurate pre-treatment procedure of biological samples was developed, using solid-phase extraction with C8 cartridges (100mg, 1mL) and needing small amounts of plasma or urine (300microL). The calibration curves were linear over a working range of 10.0-1500.0ng/mL for methadone and of 5.0-500.0ng/mL for buprenorphine and norbuprenorphine in both matrices. The limit of quantitation (LOQ) and the limit of detection (LOD) were 1.0 and 0.4ng/mL for methadone and 0.5 and 0.2ng/mL for both buprenorphine and norbuprenorphine, respectively. The method was successfully applied to the analysis of plasma and urine samples from patients undergoing treatment with these drugs. Precision and accuracy results were satisfactory and no interference from endogenous or exogenous compounds was found. The method is suitable for the simultaneous determination of methadone and buprenorphine in human plasma and urine for therapeutic drug monitoring purposes.     

LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood

Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)-ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.     

Enhanced sensitivity to methadone in adult rats perinatally exposed to methadone

Adult rats, maternally exposed to methadone during gestation and/or lactation, were evaluated for thermoregulatory and nociceptive responsiveness following a challenge with 5 mg/kg (i.p.) methadone. Prior to drug administration, female rats in the gestation and gestation-lactation groups and all male rats perinatally exposed to methadone were subnormal in body temperature. One hour after acute methadone injection, male control rats were hypothermic. All groups of methadone-treated offspring exhibited a marked lowering in body temperature with respect to their pre-injection levels, as well as in regard to values of methadone-administered control animals. Prior to drug administration, male rats of the gestation-lactation group and female rats of the gestation and lactation groups had elevated nociceptive thresholds. Except for male rats in the lactation group, animals treated with methadone perinatally had longer latencies in response to the hot-plate relative to their pre-injection values, as well as to levels of methadone-injected controls. Three days after acute methadone administration, some groups of rats subjected to this drug during gestation and/or lactation were found to be hypothermic and hypalgesic in respect to their pre-injection values, and also relative to control rats. These results suggest that exposure to methadone early in life can have a profound influence on drug response in adulthood.     

Seasonality of serum testosterone levels and sperm density in Tursiops truncatus

We trained a mature male bottlenose dolphin, Tursiops truncatus, to provide semen samples on command. After completion of the 10-week training period, semen was collected twice weekly and blood was sampled twice monthly for a period of 28 months. Total sperm per ejaculate ranged from near 0 to 54.6 x 10(9) (n = 1332). Sperm densities from each session ranged from no sperm to 1,587 x 10(6)/ml (n = 241). Testosterone levels ranged from 1.1 to 54.4 ng/ml (n = 79). Seasonal variations were observed in total sperm per ejaculate, sperm density per ml of ejaculate, and in serum testosterone levels. Peak sperm densities were detected during September and October of three consecutive breeding seasons. Serum testosterone levels peaked in June, decreased during July and August, and were lowest in September and October, the period of greatest sperm density. Peak sperm production and density were coincident with the peak period of breeding activity but at a time when serum testosterone levels were lowest.