Interaction of calcium and lead in human erythrocytes.

The interactions of calcium and lead on the human erythrocytes have been studied in vitro using 45Ca and 203Pb as tracers. The chemical groups binding calcium and lead on the erythrocytes were also investigated. Calcium ions in the plasma were shown to be capable of replacing the 203Pb on the red cells. More than 85% of the 203Pb in the erythrocyte was associated with the cytoplasmic components, and the rest was bound to the stromal membrane. About 90% of 45Ca was attached to erythrocyte membrane. Extraction of 45Ca and 203Pb-labelled erythrocyte membranes with chloroform/methanol mixture showed that the distribution patterns of these two nuclides are similar, with over 88% protein bound, less than 10% lipid bound, and traces in the aqueous phase. Chemical modification of erythrocyte membrane proteins with carbodi-imide, p-chloromercuribenzoate (PCMB), and maleic anhydride suggested that the carboxyl groups are responsible for binding lead and calcium to the red cell membrane. The SH groups may have a minor role in the binding for both cations. Amino groups did not appear to affect the binding of these cations. Gel chromatography of 45Ca-labelled erythrocyte membrane indicated that Ca++ bound to the same fraction of membrane proteins as 203Pb, corresponding to a molecular weight of about 130 000 to 230 000. A possible implication of these findings is that lead and calcium may compete for the same binding site(s) on the erythrocyte.     

Chelatable fraction of 203Pb in blood of young and adult rats

The binding of lead in the blood of young and adult rats was studied. A single intravenous dose of lead citrate (0.5 mg/kg) labeled with ²⁰³Pb was given to 2-week-old suckling rats and 19-week-old female albino rats. The whole blood content and distribution of lead between plasma and blood cells were measured 24 hr later. The chelatable fraction of lead was estimated in vitro by incubating the blood cells of young and adult rats with a combination of chelating agents—EDTA plus BAL (0.4 mmole). This experiment revealed that young rats accumulated a higher percentage of the lead dose in whole blood in comparison to adults. The results of distribution of lead between plasma and blood cells showed that young rats had a higher content of lead in plasma. The chelatable fraction of lead bound to blood cells was almost three times greater in immature animals than in adults. It is concluded that lead binding in blood basically differs between young and adult rats probably due to the existence of different lead-binding ligands.     

Lipid peroxidation and concentration of glutathione in erythrocytes from workers exposed to lead

Lipoperoxide concentration in erythrocytes from workers occupationally exposed to lead (mean blood lead concentration 57.1 (SD 17.6) micrograms/dl) was significantly higher than that in controls. It was not different in plasma from the two groups. The activity of superoxide dismutase (SOD) and catalase in erythrocytes from workers exposed to lead was significantly lower than that of control subjects. The effect of lead was also seen in the glutathione concentration of erythrocytes from lead exposed workers, which was reduced to 69% of that found in erythrocytes from control workers. The increase in methaemoglobin content of erythrocytes from workers exposed to lead was less than expected and not significantly different from that of controls. A positive correlation between lipoperoxide concentration in erythrocytes and lead concentration in blood and a negative correlation between glutathione concentration in erythrocytes and blood lead concentration were found. Incubation of erythrocytes for 24 hours at 37 degrees C in the presence of lead (100 micrograms/dl) produced no changes in glutathione and lipoperoxide concentrations, although there was inhibition of activity of SOD (14.3%), catalase (10.1%), and glutathione peroxidase (35.1%). A similar experiment with heparinised whole blood showed increased haemolysis with no changes in membrane lipid peroxidation of erythrocytes. It is postulated that the lowered concentration of glutathione and decreased activity of SOD, catalase, and glutathione peroxidase in erythrocytes from workers exposed to lead may play a part in the increased membrane lipid peroxidation. Furthermore, the results suggest the possibility that leucocytes, or platelets, or both, may induce haemolysis in the presence of lead.     

Lipid peroxidation and concentration of glutathione in erythrocytes from workers exposed to lead.

Lipoperoxide concentration in erythrocytes from workers occupationally exposed to lead (mean blood lead concentration 57.1 (SD 17.6) micrograms/dl) was significantly higher than that in controls. It was not different in plasma from the two groups. The activity of superoxide dismutase (SOD) and catalase in erythrocytes from workers exposed to lead was significantly lower than that of control subjects. The effect of lead was also seen in the glutathione concentration of erythrocytes from lead exposed workers, which was reduced to 69% of that found in erythrocytes from control workers. The increase in methaemoglobin content of erythrocytes from workers exposed to lead was less than expected and not significantly different from that of controls. A positive correlation between lipoperoxide concentration in erythrocytes and lead concentration in blood and a negative correlation between glutathione concentration in erythrocytes and blood lead concentration were found. Incubation of erythrocytes for 24 hours at 37 degrees C in the presence of lead (100 micrograms/dl) produced no changes in glutathione and lipoperoxide concentrations, although there was inhibition of activity of SOD (14.3%), catalase (10.1%), and glutathione peroxidase (35.1%). A similar experiment with heparinised whole blood showed increased haemolysis with no changes in membrane lipid peroxidation of erythrocytes. It is postulated that the lowered concentration of glutathione and decreased activity of SOD, catalase, and glutathione peroxidase in erythrocytes from workers exposed to lead may play a part in the increased membrane lipid peroxidation. Furthermore, the results suggest the possibility that leucocytes, or platelets, or both, may induce haemolysis in the presence of lead.     

High affinity of lead for fetal haemoglobin

In-vitro experiments using 203Pb were performed to identify lead-binding components in human haemoglobin. Sephadex A-50 ion-exchange chromatography of haemolysate showed that different types of haemoglobin had different affinities for lead. For the haemolysate from adults, lead was present in both Hb A (alpha 2 beta 2) and Hb A2 (alpha 2 delta 2), whereas, in the haemolysate from new-born infants, the haemoglobin of fetal origin, Hb F (alpha 2 gamma 2) showed a much greater affinity for 203Pb than the adult haemoglobin Hb A (alpha 2 beta 2), obtained from maternal blood. Analysis of the 203 Pb-labelled haemoglobin suggested that about 82% of 203Pb was in the globin polypeptide. Further analysis with carboxylmethyl (CM) cellulose chromatography indicated that the gamma globin of fetal origin had a higher affinity for 203Pb than the beta globin, whereas alpha globin appeared to be unimportant in lead binding. The results of the different affinities for lead of different Hb types are discussed with regard to the effect of lead upon haemoglobin synthesis.     

High affinity of lead for fetal haemoglobin.

In-vitro experiments using 203Pb were performed to identify lead-binding components in human haemoglobin. Sephadex A-50 ion-exchange chromatography of haemolysate showed that different types of haemoglobin had different affinities for lead. For the haemolysate from adults, lead was present in both Hb A (alpha 2 beta 2) and Hb A2 (alpha 2 delta 2), whereas, in the haemolysate from new-born infants, the haemoglobin of fetal origin, Hb F (alpha 2 gamma 2) showed a much greater affinity for 203Pb than the adult haemoglobin Hb A (alpha 2 beta 2), obtained from maternal blood. Analysis of the 203 Pb-labelled haemoglobin suggested that about 82% of 203Pb was in the globin polypeptide. Further analysis with carboxylmethyl (CM) cellulose chromatography indicated that the gamma globin of fetal origin had a higher affinity for 203Pb than the beta globin, whereas alpha globin appeared to be unimportant in lead binding. The results of the different affinities for lead of different Hb types are discussed with regard to the effect of lead upon haemoglobin synthesis.     

Hair mercury negatively correlates with calcium pump activity in human term newborns and their mothers at delivery

BACKGROUND: Calcium homeostasis is a known target of several environmental toxicants including lead and mercury. OBJECTIVE: Our goal was to determine the relationship between Hg exposure and erythrocyte Ca pump activity in women at delivery and in their newborns. METHODS: We determined total Hg as well as Pb concentrations in 81 hair and blood samples obtained at delivery. Basal and calmodulin-stimulated Ca pump activity was measured in red blood cells from cord blood and maternal erythrocyte plasma membranes. RESULTS: Maternal hair Hg negatively correlates with Ca pump activity in maternal and cord blood erythrocytes. Pb and Hg both independently correlate negatively with Ca pump activity without any statistically significant interaction. After adjustment for potential confounders, Pb and Hg explain about 30% and 7% of total variance of Ca pump activity in newborns and mothers, respectively. CONCLUSION: Our findings confirm results reported in previous experimental studies and support the use of biomarkers in newborns from general population.     

<Emphasis Type="Italic">In Vitro</Emphasis> Study of Methylmercury in Blood of Bottlenose Dolphins (<Emphasis Type="Italic">Tursiops truncatus</Emphasis>)

The biochemical behavior of methylmercury (MeHg) in dolphin blood was investigated in vitro. MeHg distribution between plasma and erythrocytes and its release from erythrocytes into plasma or medium without SH group was determined. At the subcellular level its distribution among different thiol-containing molecules was also investigated in erythrocytes and plasma. When blood was treated with 0.1 mM MeHg, about 98.1% was found in red cells and 1.9% in plasma; only 0.6% of MeHg present in the cellular compartment was bound to membranes. Hemoglobin (Hb) and albumin, principal proteins containing SH groups (PSH), and glutathione (GSH) appeared to be the main targets of MeHg in dolphin blood. Gel filtration of stroma-free hemolysate of treated red blood cells (RBCs) revealed that MeHg was almost equally present in high (52.5%) and low (47.5%) molecular weight fractions, whereas in plasma it only eluted with proteins (high molecular weight fractions). Hemoglobin was identified as the main intracellular protein binding MeHg. The exchange reaction of MeHg between GSH and dolphin hemoglobin was also evaluated and the equilibrium constants calculated. Received: 14 May 2001/Accepted: 1 October 2001     

The effect of age on the absorption and excretion of lead.

The retention (the proportion of ingested or administered lead found in the carcass at slaughter) of lead in the carcass and tissues of rats given 203Pb intraperitoneally or by mouth was measured over a period of a few days at 3-month intervals for 9 months from weaning. Total carcass retention of 203Pb administered intraperitoneally reached a minimum at 6 months of age, but the retention of 203Pb in blood, liver, and kidney changed little with time. When 203Pb was given by mouth, the fraction of the dose absorbed and the fraction retained in the carcass reached a minimum at 6 months, but the recovery in the tissues continued to decrease up to the end of the experiment. The addition of 20 mg of lead/kg of diet had no consistent effect on the metabolism of 203Pb.     

Changes of membrane fluidity in erythrocytes of lead-exposed workers

Summary We have studied the effect of lead on the fluidity of erythrocyte membrane to clarify if lead can interact in vivo with biological membranes. Erythrocyte membranes were chosen in our study because a decrease of red cell osmotic fragility is also evident in the absence of laboratory and clinical signs of anaemia. The study was undertaken using the Electron Spin Resonance technique with two spin labels 5-doxyl stearate and 16-doxyl-stearate, which probe the physical state of the polar surface and the inner core of the membrane respectively.Red blood cells and erythrocyte ghosts were prepared from the blood of workers occupationally exposed to lead and from healthy controls. The determinations of Pb blood, Pb urine, urine coproporphyrin and -amino levulinic acid showed an increased internal dose of lead, but the ordinary metabolic and haematological parameters were in the normal range. Our results show that in lead workers there is a change in chemical physical state both in erythrocytes and erythrocyte ghosts consistent with a decrease of membrane fluidity, which is evident in the surface as well as in the inner core of the membrane. The degree of membrane fluidity modification does not appear correlated with blood lead level. Changes in the membrane structural organization could be the molecular basis of some pathological alterations induced by lead.     

Determination of RBC membrane and serum lipid composition in trinidadian type II diabetics with and without nephropathy

Aim:

The rheological properties of erythrocytes are impaired in diabetes mellitus, especially because of changes in their membrane lipid composition.The aim of this study was to determine and examine the relationship between red blood cell (RBC) membrane and serum lipid composition in type II diabetes subjects with and without nephropathy.

Methods:

Trinidadian subjects aged 18–65 years were recruited for the study regardless of gender and ethnicity. Fasting blood samples were collected from 60 subjects of whom 20 were healthy individuals, 20 had type II diabetes without complications, and 20 were type II diabetics with nephropathy. Weight, height, waist/hip ratio, and blood pressure were recorded. All the blood samples were analysed to determine the serum lipid concentration, membrane lipid composition and plasma glucose concentration.

Results:

The body mass index and the systolic blood pressure of the diabetics (28.17 ± 4.98 kg/m², 153.21 ± 22.10 mmHg) and those with nephropathy (25.87 ± 4.68, 158.60 ± 22.49 mmHg) were higher when compared with controls (24.67 ± 5.18, 119.15 ± 13.03 mmHg). The diabetic (175.89 ± 102.73 μg/mgprotein) and diabetic nephropathy (358.80 ± 262.66) subjects showed significantly higher levels of RBC membrane cholesterol compared with controls (132.27 ± 66.47). The membrane phospholipids, protein and Na⁺/K⁺ATPase concentrations were altered in diabetics and diabetic nephropathy patients when compared with controls. The trends of increased serum cholesterol and decreased high-density lipoprotein in diabetics and diabetic nephropathy patients were noted as compared with controls but they are not significant as expected. The low-density lipoprotein cholesterol was significantly higher in diabetics when compared with diabetic nephropathy and control subjects.

Conclusions:

Our data suggest that there is a relationship between RBC membrane and serum lipid composition in subjects with type II diabetes with and without nephropathy. This relationship shows that diet and lifestyle plays a significant role in the alterations of the lipids both in serum and RBC membrane. The membrane and serum lipid composition may be used as possible indicators for type II diabetic patients with and without nephropathy to control their diet in the beginning stages to prevent them from further complications.     

Altered red cell membrane fluidity during schizogonic development of malarial parasites (Plasmodium falciparum and P. lophurae)

The plasma membranes of human or duckling erythrocytes infected with malarial parasites (Plasmodium falciparum and P. lophurae respectively) were stained by the fluorescent dye merocyanine 540 in the presence of serum. Unparasitized erythrocytes from infected ducklings or from in vitro cultures remained unstained in the presence of serum. Because merocyanine 540 has a greater affinity for fluid phased or disordered lipid bilayers the results suggest that upon infection of the red blood cell the erythrocyte plasma membrane becomes disordered or is increased in its fluidity. Such alterations of the host erythrocyte are probably due to parasite-induced modifications in the underlying spectrin network (required for lipid leaflet asymmetry) as well as changes in erythrocyte membrane lipid composition.     

The relationship between lead in plasma and whole blood in women

Studies have suggested that plasma lead levels may better reflect the toxicologically labile fraction of circulatory Pb that is more freely available for exchange with target tissues than do Pb levels in whole blood. Studies have also reported an apparent severalfold variation in the relative partitioning of Pb between whole blood and plasma (or serum) for a given whole-blood Pb level. This may reflect inherent differences in the plasma Pb/whole blood Pb partitioning among individuals and/or methodologic challenges associated with the collection and analyses of samples that generally contain < 1-2 ng total Pb. Here, we conducted a longitudinal assessment of the relationship between Pb in whole blood and plasma in environmentally exposed reproductive-age women (n = 63) living in Mexico City, Mexico. We collected whole blood and plasma samples using trace metal clean techniques and analyzed them for Pb using high-resolution inductively coupled plasma mass spectrometry. A subset of subjects provided repeated blood samples weekly for 4 consecutive weeks (n = 17 subjects) or every 1-2 months over a 9-month period (n = 14 subjects). Plasma Pb concentration was significantly positively associated with whole-blood Pb in a curvilinear fashion over the range of blood Pb values observed here (2.13-39.7 microg/dL). This relationship was best described by the function Plasma Pb = e (-2.392 + 0.0898 x blood Pb), where SE(coefficient) = 0.0054, SE(constant) = 0.063 (n = 63 subjects, n = 141 observations). Results from the short- and long-term repeated collection subjects indicated that the within- and between-subject variance components were not significantly different between the two subsets of subjects. The between-subjects component accounts for 78% of the variance in plasma Pb levels, while the residual variance (22%) may be attributed to other unmeasured factors. Collectively, this study demonstrates that plasma Pb measurements may be applied to general clinical settings, provided that established trace metal clean techniques are adopted. This study also shows that the relative (%) partitioning of whole-blood Pb in plasma naturally varies by a factor of about 2-4-fold among subjects at a given blood Pb level. Because Pb in the plasma is considered to more closely represent the fraction of Pb in the circulation that is readily exchanged with peripheral target tissues (e.g., brain, kidney, skeleton), the routine assessment of plasma Pb may provide a more meaningful measure of toxicologically available Pb.     

Du-zhong (Eucommia ulmoides Oliv.) cortex water extract alters heme biosynthesis and erythrocyte antioxidant defense system in lead-administered rats

This study examined the ameliorative effect of a Du-zhong (Eucommia ulmoides Oliv.) cortex water extract (DzCw) on heme biosynthesis and erythrocyte antioxidant enzyme activities in lead (Pb)-administered rats. Male rats were divided into three groups: normal control group, Pb control group (Pb), and DzCw-administered Pb group (Pb + DzCw). The Pb (25 mg/kg of body weight) was administered orally once a week for 4 weeks, while the DzCw was administered orally at a dosage of 0.139 g of DzCw/kg of body weight/day. DzCw administration significantly lowered plasma Pb concentration compared with the Pb group. Furthermore, the blood hematocrit and hemoglobin levels were significantly higher in the Pb + DzCw group than in the Pb group. Although the blood and hepatic delta-aminolevulinic acid dehydratase (ALAD) activities were significantly lower in the Pb group compared with the normal control group, both ALAD activities was normalized with the administration of DzCw. The erythrocyte superoxide dismutase and catalase activities were significantly higher in the Pb group than in the normal control group, whereas the glutathione peroxidase activity and glutathione level were lowered by Pb administration compared with the normal group. However, the administration of DzCw was found to enhance the antioxidant defense system and significantly lower lipid peroxidation levels in erythrocytes compared with the Pb group. These results indicate that the DzCw administration alleviated the Pb-induced oxidative stress in the erythrocytes through elevating the blood and hepatic ALAD activity and enhancing the antioxidant enzyme activities.     

Pyruvate Kinase Activity and δ-Aminolevulinic Acid Dehydratase Activity as Biomarkers of Toxicity in Workers Exposed to Lead

Lead (Pb(2+)) is a heavy metal that has long been used by humans for a wide range of technological purposes, which is the main reason for its current widespread distribution. Pb(2+) is thought to enter erythrocytes through anion exchange and to remain in the cell by binding to thiol groups. Pyruvate kinase (PK) is a thiol-containing enzyme that plays a key role in erythrocyte cellular energy homeostasis. δ-aminolevulinic acid dehydratase (δ-ALAD) is the second enzyme in the heme biosynthetic pathway and plays a role in the pathogenesis of Pb poisoning. Our primary objective was to investigate the effect of Pb(2+) on the activity of the thiolenzymes δ-ALAD and PK and on the concentration of glutathione (GSH), a nonenzymatic antioxidant defense, in erythrocytes from Pb-exposed workers. The study sample comprised 22 male Pb workers and 21 normal volunteers (15 men and 6 women). The Pb-exposed workers were employed in manufacturing and recycling of automotive batteries. Basic red-cell parameters were assayed and total white blood cell counts performed. PK and δ-ALAD activity and blood Pb (BPb) concentrations were determined in all subjects. Pb-exposed individuals had significantly greater BPb levels than controls. Both PK and δ-ALAD activity levels were significantly lower in Pb-exposed individuals than in controls. Pb significantly inhibited PK and δ-ALAD activity in a dose-dependent manner. We found that erythrocyte GSH levels were lower in Pb-exposed individuals than normal volunteers. Pb-exposed individuals had lower values than controls for several red cell parameters (hemoglobin, hematocrit, red blood cell count, mean corpuscular volume). These results suggest that Pb inhibits δ-ALAD and PK activity by interacting with their thiol groups. It is therefore possible that Pb disrupts energy homeostasis and may be linked with decreased glucose metabolism because it affects the heme synthesis pathway in erythrocytes, contributing to the cell dysfunction observed in these in Pb-exposed individuals. These results indicate an apparent dose-effect relationship between PK activity and BPb. PK activity in human erythrocytes can be used for biological monitoring of Pb exposure. Study of the mechanisms by which Pb acts may contribute to greater understanding of the symptoms caused by Pb.     

The fate of lead in petrol-engine exhaust particulates inhaled by the rat.

Tetraethyl lead, labelled with ²⁰³Pb, was added to lead-free petrol which was used to fuel a four-stroke engine. The exhaust aerosol, after reduction of the CO concentration, was inhaled by rats using a ‘nose-only’ technique. Following exposure, animals were sacrificed serially over a period of 8 days and the ²⁰³Pb content of the various tissues and excreta was measured radiometrically. The interpretation of the results was complicated by the ingestion of ²⁰³Pb in particulates deposited on the pelt during exposure. It was established in separate experiments, however, that ²⁰³Pb in ingested exhaust particulates is not absorbed from the gastrointestinal tract (GI tract) to a significant extent. It was possible to show that 20–25% of the inhaled ²⁰³Pb was deposited in the respiratory tract, mainly in the alveolar region of the lungs. At least half of the ²⁰³Pb deposited in the lungs was absorbed with a half-time of less than 1 hour. After a week only about 2% of the deposited activity remained. The metabolism of absorbed lead is similar to that observed following its intravenous injection in chloride form. While the rate of absorption of ²⁰³Pb from particulates deposited in the lungs appears to be rather similar in rat and in man, the metabolism of absorbed ²⁰³Pb shows marked differences.     

Horse meat consumption affects iron status,lipid profile and fatty acid composition of red blood cells in healthy volunteers

This study investigated the effect of moderate consumption of horse meat on iron status, lipid profile and fatty acid composition of red blood cells in healthy male volunteers. Fifty-two subjects were randomly assigned to two groups of 26 subjects each: a test group consuming two portions of 175 g/week of horse meat, and a control group that abstained from eating horse meat during the 90 days trial. Before and after 90 days, blood samples were collected for analysis. Horse meat consumption significantly (p ≤ 0.05) reduced serum levels of total and low-density lipoprotein cholesterol ( ? 6.2% and ? 9.1%, respectively) and transferrin ( ? 4.6%). Total n ? 3, long chain polyunsaturated fatty acids n ? 3 and docosahexeanoic acid content in erythrocytes increased (p ≤ 0.05) by about 7.8%, 8% and 11%, respectively. In conclusion, the regular consumption of horse meat may contribute to the dietary intake of n ? 3 polyunsaturated fatty acids and may improve lipid profile and iron status in healthy subjects.     

The effect of maternal dose on lead retention in suckling rats

Lead (Pb) transfer from mother to litter was investigated at the late stage of lactation after a single intrapitoneal injection of 2.0 microgram Pb/ml marked with 203Pb. After 48 hr almost 20% of the maternal dose of 203 Pb was found in the litter, and about 0.6 and 0.2 of the injected dose was found in the liver and kidneys of suckling rats, respectively. Similar whole-body retention was observed earlier in suckling rats after a 20 times lower dose of stable lead was injected intravenously into their mothers.     

The Effect of Maternal Dose on Lead Retention in Suckling Rats

Lead (Pb) transfer from mother to litter was investigated at the late stage of lactation after a single intraperitoneal injection of 2.0 μg Pb/ml marked with ²⁰³Pb . After 48 hr almost 20% of the maternal dose of ²⁰³Pb was found in the litter, and about 0.6 and 0.2% of the injected dose was found in the liver and kidneys of suckling rats, respectively. Similar whole-body retention was observed earlier in suckling rats after a 20 times lower dose of stable lead was injected intravenously into their mothers.     

Disposition and excretion of 2-chlorodibenzofuran in the rat

When 2-chloro[¹⁴C]dibenzofuran was intravenously administered to the rats, about 86% of radioactivity was found in the urine, the large intestinal contents and the feces within 24 h. Approximately 3% of 2-chlorodibenzofuran (2CDF) radioactivity was present in the adipose tissue 48 h after an intravenous administration. A similar excretion pattern of the compound was observed in orally administered rats. From experiments in vitro, approximately 80% of 2CDF was present in the red blood cell fraction and the remainder in the plasma fraction. Bile cannulation studies revealed involvement of enterohepatic circulation in the metabolism of the compound. Bile specimens that were subjected to enzymatic hydrolysis by arylsulfatase and/or -glucuronidase showed the presence of various yet unidentified conjugated substances. A comparative study using 3-chlorodibenzofuran (3CDF) showed a similar distribution pattern in the 2CDF-treated rats, but 2CDF appeared to accumulate more in the adipose tissue and red blood cells. The present study demonstrates that most of 2CDF is quickly metabolized to give rise to a number of metabolites and excreted from the body, but suggests that the compound once distributed in the adipose tissue may remain there for a relatively long period of time.