Lead toxicity and chelation therapy.

PURPOSE: Common sources of lead exposure, the primary clinical effects of lead toxicity, and current recommendations for managing lead toxicity, including chelation therapy, are reviewed. SUMMARY: Common sources of lead exposure in children and adults include industrial and mining activities, paint, dust, soil, water, air, the workplace, food, trinkets, ethnic folk remedies, and cosmetics. The absorption and biological fate of lead are affected by a variety of factors, including an individual's nutritional status, health, and age. Children with a blood lead concentration of >10 microg/dL and adults with a blood lead concentration of > or = 45 mug/dL should undergo further evaluation. Symptoms and time to onset of symptoms postexposure may vary, and it can be difficult to identify the early, subtle neurologic effects of lead toxicity. The classic symptoms of lead toxicity generally correlate with blood lead concentrations of 25-50 microg/dL in children and 40-60 microg/dL in adults. Management of lead toxicity requires extensive risk assessment and caregiver education. Chelation is generally not indicated for adults with blood lead concentrations of < 45 microg/dL because of the potential risk of adverse drug events and concerns about remobilized lead, and chelation for children with blood lead concentrations of < 45 microg/dL remains controversial. Dimercaprol, edetate calcium disodium, and succimer are the three agents primarily used for chelation. CONCLUSION: Lead toxicity remains a significant public health concern. Elimination of elevated blood lead levels in children can be accomplished by educating appropriate health care providers and caregivers, recognizing potential lead sources, and adopting aggressive prevention and case management measures.     

Safety and efficacy of meso-2,3-dimercaptosuccinic acid (DMSA) in children with elevated blood lead concentrations

OBJECTIVE: To evaluate the safety and efficacy of meso-2,3-dimercaptosuccinic acid in the treatment of children with lead toxicity. DESIGN: This was an open-label study in 59 children 12-65-months old, with pretreatment whole-blood lead levels of 25-66 microg/dL, who received 116, 26-28 day courses of oral dimer-captosuccinic acid, while residing either in the Pediatric Clinical Research Unit of the Johns Hopkins Hospital or in lead-safe housing during the outpatient portion of the study. RESULTS: All, who completed the study, showed sharp decreases in blood lead concentration during therapy, but 2-3 weeks following completion of drug therapy, blood lead concentration rebounded to an average of 58% (23 microg Pb/dL of whole blood) of their average pretreatment blood lead concentration (40 microg Pb/dL of whole blood). There were no adverse reactions attributable to dimercaptosuccinic acid; however, 2 of the 59 patients were reexposed to defective lead paint and experienced sharp increases in blood lead concentration while on therapy. In one instance, the child's blood lead concentration increased from 20 to 90 microg Pb/dL whole blood in 1 week. Other unexpected events were discussed in the text. CONCLUSIONS: Dimercaptosuccinic acid is apparently safe and does mobilize lead into the urine, but not the essential metals, zinc and copper. Reexposure is always a danger; therefore, all children, while on therapy, should be monitored for their blood lead concentration at weekly intervals during and immediately after therapy. No conclusions can be drawn from this study regarding long-term beneficial effects, if any, of this drug on late neurocognitive outcome.     

Acute elevation of blood lead levels within hours of ingestion of large quantities of lead shot

BACKGROUND: Ingestion of elemental lead foreign bodies is felt to have a low risk of clinically significant lead absorption unless gastrointestinal pathology and/or prolonged transit time are present. We present a case of ingestion of a large quantity of small diameter lead shot accompanied by rapid elevation of blood lead levels. CASE REPORT: A 5 1/2-year-old previously healthy girl was found eating the pellets from an ankle weight. She vomited and complained of abdominal pain. In the emergency department, she had no complaints and normal vital signs. An abdominal X-ray showed thousands of small, round, metallic density objects in the stomach. Her white blood cell count was 14,700/mm3, and the hemoglobin, mean corpuscular volume, free erythrocyte protoporphyrin, zinc protoporphyrin, biochemistry panel 21, and urinalysis were normal. She had no prior lead level for comparison. Whole-bowel irrigation was begun and she passed over 11 stools with pellets as well as other foreign bodies (erasers, bead, etc.) in the first 24 hours. Pellets were still seen on X-ray the following day so she received a high-fiber diet and bisacodyl tablets 10 mg/d. On hospital day 2, her admission blood lead (drawn 13 hours after ingestion) was reported as 57 microg/dL (2.7 microm/L) and chelation was begun with oral 2,3-dimercaptosuccinic acid 10 mg/kg 3x/d for 5 days, then 2x/d for 14 days. Her peak measured lead level was 79 microg/dL approximately 36 hours after ingestion. She excreted 2,273 microg lead in the urine during her first 24 hours of chelation. Her blood lead dropped to 14.3 microg/dL by the end of chelation. She did not develop any apparent signs of lead poisoning. CONCLUSION: Acute elevations of blood lead concentrations may occur rapidly after ingestion of multiple small elemental lead objects.     

Should all patients with unexplained anaemia be screened for chronic lead poisoning?

The global prevalence of lead poisoning is declining. However, the prevalence of lead poisoning in patients with either microcytic or normocytic anaemia is unknown. Blood samples from anaemic patients residing in south-east London without an obvious cause for anaemia had their blood lead concentration (BLC) analysed. A batch of 988 samples was analysed for BLC using atomic absorption spectroscopy. Median haemoglobin was 10.3 g/dL (range: 4.2-10.9) in females, 10.6 g/dL (range: 5.2-11.4) in males and 10.7 g/dL (range: 6.7-10.9) in children. Median BLC was 2.63 microg/dL (0.21-24.0 microg/dL; 95th centile 7.54 microg/dL). Fifteen samples (1.5%) had a BLC > 10.0 microg/dL, five samples (1%) > 15.0 microg/dL and one sample (0.1%) > 20.0 micrg/L. In the 106 children, median BLC was 2.34 microg/dL (0.5-14.5 microg/dL; 95th centile 6.12 microg/ dL). Only one child (14.5 microg/L) had a BLC > 10.0 pg/dL. There was a poor correlation between haemoglobin and BLC (r2 = 0.08). Routine screening for lead poisoning cannot be justified in all patients with unexplained anaemia, unless there is a history or clinical features to suggest lead toxicity. Additionally, we have shown that in this former high-risk area for lead exposure, there is a low point prevalence of significant lead poisoning, even in an anaemic population.     

Delta-aminolevulinic acid dehydratase inhibition and oxidative stress in relation to blood lead among urban adolescents

To explore lead-induced oxidative stress among urban adolescents, the present study, the first from India, was designed to determine the proportion of urban adolescents with blood lead >10 microg/dL and its impact on selected oxidative stress parameters and delta-aminolevulinic acid dehydratase (delta-ALAD) inhibition, which could be used as biomarkers of lead intoxication. A total of 39, urban, male adolescents, drawn from Lucknow and adjoining areas, were recruited to determine lead, delta-ALAD, malondialdehyde (MDA) and glutathione (GSH) in blood and catalase (CAT) in RBCs. Mean level of blood lead was 9.96 +/- 3.63 microg/dL (4.62-18.64); 43% of adolescents crossed the Centre for Disease Control (CDC) intervention level of 10 pg/dL blood lead. On the basis of blood lead levels (BLLs), adolescents were categorized into two groups: Group I and Group II had a blood lead <10 microg/dL (7.40 +/- 1.62) and >10 microg/dL (13.27 +/- 2.67), respectively, with significantly different mean values (P <0.001). Age, sex, body mass index (BMI), Hb level (malnutrition), and area of living as confounders of lead exposure and toxicity were not statistically different between the two groups. However, delta-ALAD activity was significantly lower (P <0.001), while CAT activity was higher in Group II than in Group I (P <0.01). MDA level was also significantly higher in Group II compared to Group I (P <0.001). There were significant negative correlation of BLL with 6-ALAD (r= -0.592, P <0.001), and positive correlations with CAT (r=0.485, P <0.01) and MDA (r=0.717, P <0.001). Interestingly, delta-ALAD, in turn, had significant negative correlations with CAT (r= -0.456, P <0.01) and MDA (r= -0.507, P <0.01). Results of the present pilot study provide clues to the possible low level of lead-induced oxidative stress in urban adolescents, suggesting that lead-induced 6-ALAD inhibition can also be an indicator of oxidative stress. The potential of oxidative stress parameters to be used as biomarkers of lead toxicity warranted further investigation.     

Lead contaminated moonshine: a report of Bureau of Alcohol, Tobacco and Firearms analyzed samples

The CDC's Healthy People 2010 has set a US population lead level goal of < 25 microg/dL. A recent study of Emergency Department patients in Atlanta, GA, revealed a significant association between reported moonshine consumption and elevated blood lead. However, beyond anecdotal reports and isolated case histories, laboratory analyses confirming the presence and extent of lead contamination among moonshine samples are absent from modern scientific literature. One hundred and fifteen suspected moonshine samples seized by local law enforcement between 1995 and 2001 were voluntarily submitted to the Bureau of Alcohol, Tobacco and Firearms' National Laboratory for lead analysis using flameless atomic absorption spectrophometry. Samples originated from 9 states: 5 southeastern states, Missouri, Ohio, Wisconsin and West Virginia. Lead levels ranged between 0.0 microg/dL and 53,200 microg/dL (median 44.0 microg/dL). Median percent alcohol by volume was 44.75% (range 3.85-65.80%). Thirty-three samples (28.7%) contained lead levels > 300 microg/dL, the limit designated potentially hazardous by the FDA. Percent alcohol by volume did not predict lead content. Consuming 1 L/d of moonshine contaminated with 400 microg/dL of lead would result in a blood lead level of approximately 25 microg/dL. At a high level of consumption, 25% of the samples could produce blood lead levels > or = 25 microg/dL. Moonshine production and consumption is an under-appreciated toxicologic and public health concern and is not restricted to the southeastern US.     

Lead poisoning and chelation in a mother-neonate pair.

We report the case of a pregnant woman with chronic lead toxicity and a blood lead of 57 microg/dL (2.7 micromol/L) who gave birth to a healthy-appearing neonate with a cord blood lead of 126 microg/dL (6.08 micromol/L). The mother was prescribed a single course of oral succimer late in the third trimester of pregnancy, without any appreciable change in her blood lead. The neonate was initially treated with intramuscular dimercaprol and intravenous edetate calcium disodium. After 3 days, the neonate was then switched to oral 2,3-dimercaptosuccinic acid because the blood lead had declined. The child received two 19-day courses of 2,3-dimercaptosuccinic acid and had a blood lead level of 21.5 microg/dL (1.04 micromol/L) at 5 months of age. Despite extensive investigation, the precise source of the mother's lead toxicity remained undetermined.     

Effect of early lead exposure on the maturation of children's postural balance: a longitudinal study

This prospective study investigated the impact of early exposure to lead on the maturation of children's postural balance. The effect of lead exposure on age-associated maturation of postural balance was investigated on 91 children from the Cincinnati Lead Study (CLS) with a 5-year geometric mean lead concentration in blood of 11.66 microg/dL (range 3.89-28.33 microg/dL) by re-assessing their postural balance approximately every 20 months starting at mean age of 6.6 years through mean age of 12.1 years. The results presented in this paper provide evidence that low to moderate lead exposure in early childhood has a measurable and statistically significant impact on the maturation of postural balance. In comparison to less exposed children, of those in the higher lead group showed an impaired postural balance response. The results from this study suggest that children with early childhood lead exposure may need additional time to approach (or "catch up" with) their maturational postural balance status. As these subjects are now adults in their early to mid-twenties, poor postural balance may impact their daily living tasks and pose a higher risk of potential injuries at home and work.     

Blood lead and cadmium in dogs from urban India

Concentrations of lead and cadmium were estimated in blood samples of 43 healthy dogs from urban India. Concentrations were 19.5+/-2.1 microg lead/dL and 0.74+/-0.13 microg cadmium/dL, which were considerably higher for rural dogs. Sex, age, and diet had no significant effect on lead and cadmium blood concentrations, yet male and adult dogs had higher levels than females and young dogs. The use of dogs as sentinels for environment quality in India was indicated by this study.     

High levels of blood lead in griffon vultures (Gyps fulvus) from Cazorla Natural Park (southern Spain)

The blood lead of 23 griffon vultures (Gyps fulvus) trapped in 2003 was analyzed in order to evaluate exposure to lead in the vulture population of Cazorla Natural Park (in southern Spain). In 2001 the use of leaded gasoline in vehicles was banned in the European Union; however, lead ammunition is still used in Spain in big-game hunting for red deer, fallow deer, mouflon, and wild boar, which are ingested by vultures from September to March. The mean concentration of lead in blood was 43.07 +/- 31.96 microg/dL with a range of 17.39-144.80 microg/dL. Only two vultures had lead levels below 20 microg/dL, and two others had blood lead concentrations close to 150 microg/dL. In view of the results, we think the population of vultures from Cazorla Natural Park is suffering subclinical exposure to lead, with some individuals exposed to high toxicity risk. We concluded that ingestion of lead in the metallic form alone is sufficient to produce these blood lead concentrations, and we recommend the prohibition of lead ammunition for big-game hunting in order to preserve the vulture population.     

Recall of a lead-contaminated vitamin and mineral supplement in a clinical trial

PURPOSE: The Treatment of Lead-exposed Children (TLC) trial tested whether developmental outcome differed between children treated for lead poisoning with succimer or placebo. On 7 July 1997, TLC was informed that the vitamin and mineral supplements it gave to all children were contaminated with about 35 microg of lead per tablet. METHODS: TLC recalled the contaminated supplements and measured the children's exposure. RESULTS: The families of 96% of the children were contacted with 30 days. Among the 571 children to whom the contaminated supplements were dispensed, the mean increase in blood lead was 0.06+/-0.01 micromol/L (1.2+/-0.2 microg/dL); among 78 children to whom they were not, it was 0.09+/-0.03 micromol/L (1.8+/-0.7 microg/dL). There was no evidence of a dose-response relation between estimated supplement consumption and increase in blood lead concentration. CONCLUSIONS: The children's blood lead concentrations were not detectably affected by the contamination. Since the association of cognitive delay with lead exposure is best described for blood lead, we believe that the trial's inference about the effect of drug therapy on lead induced cognitive delay should be unaffected.     

Effect of lead exposure on serum immunoglobulins and reactive nitrogen and oxygen intermediate

Metal toxicants may affect immune regulation with an increased incidence of infectious diseases, cancer or autoimmune diseases. Lead is the leading environmental toxin among heavy metals and has aroused concern, as continuous low-level exposure leads to a variety of health problems. We compared serum immunoglobulins (Ig) and reactive oxygen and nitrogen intermediates (super oxide and nitric oxide (NO)) in culture supernatant of lead-exposed (blood lead; Pb-B > 10 microg/dL) individuals with that of unexposed healthy controls (blood lead < 10 microg/dL). The serum IgA level was significantly increased in lead-exposed individuals in comparison to controls (182 +/- 53 versus 138 +/- 52 mg/dL; P < 0.05). Furthermore, lipopolysaccharide-induced NO production by mouse macrophage cells, RAW 264.7, showed significant suppression (P < 0.05) after treatment with lead acetate (100 ppm). This study suggested that lead could modulate the immune system by targeting the humoral as well as innate immune cells.     

Lipids, lipid peroxidation and 7-ketocholesterol in workers exposed to lead

The study population included healthy men and hypertensive employees of zinc and lead steelworks in the south of Poland. Workers exposed to lead (n=137) were divided into two groups: the first included employees with low exposure to lead (LL) with mean blood lead (PbB) 25-40 microg/dL and the second one with PbB over 40 microg/dL (HL group). The administration workers (n=35) were the control group. Evaluation of lipids and oxidative changes of cholesterol and lipids were estimated in blood samples. No significant changes in concentration of 7-ketocholesterol and blood lipids (cholesterol, HDL, LDL, triglycerides) were found. Lipid peroxidation (LP) was significantly higher in both exposed groups in plasma and in the HL group in erythrocytes when compared with control. There can be two independent sources of LP increase: the first is connected with the direct effect of lead's ions on erythrocytes, the second is the prooxidative effect of delta-aminolevulinic acid. Hypertension in the HL group when compared with people with PbB below 40 microg/dL (OR 4.4, 95%CI 1.4-14.5) was found more often. LP significantly increased by about 71% and concentration of 7-ketocholesterol by about 122% in hypertensives when compared with normotensives in the HL group.     

Blood lead levels and specific attention effects in young children

The detrimental effects of early exposure to lead are credible and persistent, but there is presently no agreement on a safe threshold for circulating lead levels. Although several research groups have found significantly poorer cognitive performance in children who have whole blood levels as low as 5 microg/dL, most government agencies, including the EPA and the CDC, continue to use 10 microg/dL as the criterion for concern in public health advisories. Prior research has consistently indicated a negative relation between lead levels and attention. Similarly, the results of the present study show a relation between blood lead level and neurobehavioral outcome in 7-year-old children (N=506). Higher lead levels were associated significantly with decreased scores on measures of intelligence (i.e., overall, performance and verbal IQ), lengthened reaction time, hyperactivity, and social and delinquent behavior problems. Importantly, the present study documents a significant negative impact of blood lead levels on attention, but not impulsivity, in early elementary age children, further delineating the specific aspects of attention related to blood lead concentrations. Analyses were also conducted to identify a "safe" blood lead level threshold. Visual inspection of non-parametric regression plots suggested a gradual linear dose-response relationship for each endpoint. None of the neurobehavioral outcomes assessed showed evidence of a threshold under which lead levels appear to "safe". In light of the consistency of these findings with those of several other groups, it is advisable to consider whether the threshold for an acceptable blood lead level should be reduced.     

Brainstem auditory evoked response at five years and prenatal and postnatal blood lead

Previous work from this laboratory demonstrated an association between higher maternal blood lead level at 20 weeks of pregnancy and increased I-V and III-V interpeak intervals in the brainstem auditory evoked response (BAER) recorded in 1-month-old infants. We repeated the BAER measurements with a larger group of children (n = 100-113) from the same study at 5-7 years. Maternal blood lead level at 20 weeks of pregnancy (geometric mean = 7.7 microg/dl; range 1-30. 5 microg/dl) was the only prenatal blood lead level significantly associated with I-V and III-V interpeak interval in a multiple regression model controlling for head circumference and age at time of testing and sex. In contrast to the findings at 1 month of age, interpeak intervals decreased as a linear function of increasing 20-week maternal blood lead. A nonlinear, orthogonal, second-order polynomial model was a significantly better fit to the data than the linear model. The nonlinear model showed I-V and III-V interpeak intervals decreased as blood lead rose from 1 to 8 microg/dl, and then increased as blood lead rose from 8 to 30.5 microg/dl. We hypothesized that the negative linear term was related to lead effect on brainstem auditory pathway length, and that the positive quadratic term was related to neurotoxic lead effect on synaptic transmission or conduction velocity. We found support for the brainstem length interpretation in the data, showing that 6-year-old head circumference in these children significantly decreased with increased maternal 20-week blood lead level. Increasing postnatal blood lead at 12 and 48 months was related only to decreased BAER conduction intervals across the entire blood lead range, suggesting only pathway length effects. Alterations in BAER at this age may indicate that the effect of prenatal lead exposure on the auditory brainstem is permanent, as response latencies reach essentially adult values by 4 years of age.     

Prenatal and early childhood blood lead levels and cardiovascular functioning in 9(1/2) year old children

A number of studies have found that increasing lead exposure is associated with increases in blood pressure in humans. Studies with animals suggest that lead-induced increases in vascular resistance account for these increases in blood pressure. The present study assessed cardiovascular functioning at rest and in response to acute stress for 9(1/2) year old children (N=122) having relatively low prenatal (cord) blood lead levels (M=1.98 microg/dL, SD=1.75) and low postnatal (early childhood) blood lead levels (M=4.62 microg/dL, SD=2.51). Higher cord blood levels were associated with higher baseline systolic blood pressure (SBP), and higher early childhood lead levels were associated with greater total peripheral (vascular) resistance (TPR) responses to acute stress. In addition, a negative association between blood lead levels and stroke volume (SV) suggests that lead-induced increases in vascular resistance were sufficient to produce cardiac afterload, a situation arising when blood pressure in the aorta makes it difficult for the left ventricle to eject blood. These effects were not mediated by differences in task performance or emotional responses to the acute stress tasks. Finally, these effects were significant for lead levels considered low, notably, below the 10 microg/dL threshold currently adopted by the CDC for deleterious effects.     

Insulin infusion protocol for critical care units.

PURPOSE: An insulin infusion protocol for critical care units is described. SUMMARY: Evidence that aggressive glycemic control improves outcomes led physicians, nurses, dietitians, and pharmacists at a trauma center to develop an insulin infusion protocol. Before the protocol, elevated blood glucose concentrations were often not treated until they reached 200 mg/dL or higher. Insulin infusions were underutilized and were often not started until capillary blood glucose concentrations were greater than 350 mg/dL for 12 or more hours. When orders for an insulin infusion were written, they did not include directions for dosage adjustment, and the goal blood glucose range varied. A preliminary protocol was drafted allowing adjustments in insulin administration to be based on changes in capillary blood glucose values since the previous blood glucose measurement. The protocol was presented to a multidisciplinary team and further refined. The targeted blood glucose concentration range was 80-130 mg/dL. After the targeted range was achieved for a patient, if the blood glucose level continued to decrease over three consecutive measurements, the infusion rate was decreased by 0.5 or 1 unit/hr, depending on the capillary blood glucose level. Data for the first 30 patients were collected from September 2003 to August 2004. It took 2-36 hours (mean, 12.6 hours) to bring the capillary blood glucose concentration to less than 130 mg/dL. Among 2,845 capillary blood glucose measurements, there were 15 cases of hypoglycemia (0.4%) requiring treatment with 50% dextrose injection. CONCLUSION: A multidisciplinary effort resulted in the development of an insulin infusion protocol for use in critical care units.     

Elevated blood lead levels resulting from the ingestion of air rifle pellets

There have been numerous reports of lead poisoning resulting from the ingestion of foreign bodies. A case involving the ingestion of spent air rifle pellets is described. No clinical symptoms were observed, despite the fact that the young child exhibited elevated blood lead levels as high as 2.7 micromol/L (56 microg/dL). X-rays of the child's abdomen confirmed the ingestion of the pellets. The patient was treated with laxatives, and the pellets were successfully passed over the course of the next few days. Prior to release from the hospital, the child's blood lead level had dropped to 1.7 umol/L (35 microg/dL).     

The relationship between lead exposure, motor function and behaviour in Inuit preschool children

The main focus of this study was to determine the role of behaviour in the relationship between postnatal lead exposure and motor function. The sample consisted of 110 preschoolers, of age 5, from Nunavik. Lead concentration was measured at birth and at testing time. Average lead levels were of 4.9 microg/dL (0.24 micromol/L) and 5.3 microg/dL (0.26 micromol/L) for cord and child blood, respectively. Children's balance and fine motor capacities were tested. A modified version of the IBR was used to assess behaviour. Postnatal blood lead concentrations correlated positively with both impulsivity and activity. Neither pre- nor postnatal blood lead concentration correlated with attention level. The children's scores on impulsivity (I) and activity (A) were summed to create the independent variable IA, which was tested as a potential mediator between lead exposure and two dependent variables: the coefficient of covariation in alternating hand movements and transversal sway in tandem position. Mediation was significant only for the latter variable. IA and attention were then tested as potential moderators in the relation between postnatal lead exposure and motor function. No significant interaction between independent variables could be observed. These results do not support the hypothesis that, at low levels of postnatal exposure, lead acts indirectly on motor function via behaviour. However, IA does act as a mediator in the relationship between postnatal blood lead concentration and transversal sway in tandem position.     

Below background levels of blood lead impact cytokine levels in male and female mice

A number of studies have documented that Pb exerts immunotoxic effects on T lymphocytes. In studies designed to explore this general response over a broad dose range, female Swiss mice were administered six different diets containing Pb acetate 1 day after mating. During lactation, the mothers received the same feed given during pregnancy, and the same diets were administered to the offspring for 9 months after weaning. At the end of exposure, blood Pb level in the offspring was determined, and possible changes in two type 1 cytokines (IL-2, INF-gamma) and one type 2 cytokine (IL-4) in the serum were measured. At higher dietary Pb levels (40 and 400 ppm), a significant increase in IL-4 production was associated with a profound decrease in INF-gamma and IL-2 production. At the lowest Pb diet level (0.02 ppm), which resulted in a blood lead level of (0.8 microg/dL), which is below background (2-3 microg/dL) values in humans, increases in INF-gamma and IL-2 production along with a significant decrease in IL-4 production were observed. The findings provide evidence of a reversal of lead-induced cytokine skewing depending on the blood lead concentration. As blood lead concentration increases, there is a notable skewing toward Th2, while the pattern is reversed favoring Th1 development at lower blood lead values. The present findings are also notable since they indicate the potential for dietary Pb to have significant biological effects below normal background concentrations.