Field application of a colorimetric method of assaying chloroquine and desethylchloroquine in urine

In a study in western Kenya of malaria-infected adult women who had been treated with chloroquine, we compared the level of chloroquine and its principal metabolite, desethylchloroquine, in urine, measured using a newly developed modified Haskins test, with the level of chloroquine in whole blood, determined by high-performance liquid chromatography. Over a 28-day follow-up period, 277 matched urine and blood samples from 81 women were evaluated. A high correlation was observed between the level of chloroquine in whole blood (in μg/l) and that of chloroquine + desethylchloroquine in urine (in mg/l). The test was easily performed and may be useful for monitoring use of chloroquine in a community and determining pre-study or post-treatment ingestion or absorption of the drug in in vivo studies of parasite sensitivity.     

Determination of chloroquine and its metabolites in urine: a field method based on ion-pair extraction

A new straightforward photometric method for the assay of the antimalarial drug chloroquine and its metabolites in urine is described. The method involves an ion-pair extraction procedure with dichloromethane using the acid-base indicator bromthymol blue as counter-ion. The ion pair formed with chloroquine in the organic phase is yellow, and absorbance is measured at λ = 410 nm using a filter photometer. The absorbance is a linear function of concentration up to 400 μmol/l (120 mg/l) chloroquine. The method is suitable for the determination of chloroquine and its metabolites in urine down to a limiting concentration of about 10 μmol/l (3 mg/l). Additionally, the method is suitable for semiquantitative visual estimation of the concentration of chloroquine in urine. A single dose of 5 mg/kg chloroquine base could be determined in urine from two volunteers for at least 8 days after administration of the drug. The results obtained for the analysis of chloroquine and its metabolites with the colorimetric method described here correlate well with those obtained using high performance liquid chromatography.     

Increased accumulation of chloroquine and desethylchloroquine in homozygous sickle cells

The effect of haemoglobin genotype on the level of chloroquine in the erythrocytes of homozygous sickle-cell (SS), normal (AA), and heterozygous (AS) subjects was investigated in vivo and in vitro. Two hours after a single oral dose of chloroquine its level in plasma was consistently lower in SS than in AA subjects. In contrast, its level in the erythrocytes was higher in SS than in AA subjects. Desethylchloroquine, a metabolite of chloroquine, was detected only in the erythrocytes of SS blood but was present in both the plasma and erythrocytes of AA blood. For the in vitro test, a 5% suspension of erythrocytes was incubated for 1 hour with a 2.06 μmol/l solution of chloroquine. The mean chloroquine distribution ratio (μmol chloroquine per kg erythrocytes:μmol chloroquine per litre medium) was 31.0, 3.5, and 2.7 for SS, AA, and AS erythrocytes, respectively. The results of the study indicate that haemoglobin genotype appears to influence the level of chloroquine in erythrocytes.     

Adaptations of the Saker-Solomons test: simple, reliable colorimetric field assays for chloroquine and its metabolites in urine

Two field-adapted colorimetric methods for measuring the antimalarial drug chloroquine in urine are described. Both are modifications of the method of Saker and Solomons for screening urine for phencyclidine and other drugs of abuse, using the colour reagent tetrabromophenolphthalein ethyl ester. One method is semiquantitative, detecting the presence of chloroquine (Cq) and its metabolites in urine with a 1 microgram/ml detection limit; it is more sensitive and reliable than the commonly used Dill-Glazko method and is as easy to apply in the field. The second method uses a hand-held, battery-operated filter photometer to quantify Cq and its metabolites with a 2 microgram/ml detection limit and a linear range up to 8 micrograms/ml. The first method was validated in the field using a published quantitative colorimetric method and samples from a malaria study in Nigeria. The second method was validated in the laboratory against high-performance liquid chromatographic results on paired samples from the Nigerian study. Both methods may be used in remote locations where malaria is endemic and no electricity is available.     

Plasmodium falciparum: mefloquine resistance produced in vitro

Camp and Smith strains of the human malaria parasite Plasmodium falciparum became resistant to mefloquine after continuous cultivation in the presence of the drug. The 50% inhibitory dose (ID₅₀) values for mefloquine, as assessed by [³H]hypoxanthine incorporation, were found to have increased 4-fold, from 3 μg/l to 12 μg/l. The ID₅₀ values obtained by morphological examination of the cultures increased 10-fold. Resistance was stable in both strains either when grown in a drug-free medium or when kept frozen in liquid nitrogen. The mefloquine-resistant Camp strain remained sensitive to chloroquine and amodiaquine, and became slightly more resistant to quinine; there was increased sensitivity to pyrimethamine. The mefloquine-resistant Smith strain remained sensitive to amodiaquine and resistant to pyrimethamine; there was increased resistance to quinine, and an increase in sensitivity to chloroquine.     

三手烟对小鼠内皮祖细胞水平的影响及可能机制

目的探讨三手烟对小鼠内皮祖细胞水平的影响及可能机制.方法 40只8周龄清洁级雄性小鼠,分为3组并做如下处理:对照组(n=10):暴露于正常空气中;一手烟组(n=15):每日接受香烟烟雾8小时;三手烟组(n=15):每日生活在香烟烟雾熏过的无菌敷料中8小时(无菌敷料接受同一手烟组一样的香烟烟雾烟熏8小时).两个月后采血,测定并比较两组内皮祖细胞(endothelial progenitor cells ,EPCs),髓过氧化物酶(myeloperoxidase ,MPO),白细胞介素-6(interleukin -6 ,IL-6),肿瘤坏死因子-α(tumor necrosis factor -α,TNF-α),一氧化氮(nitric oxide ,NO)水平.光镜下观察比较心肌组织学变化.结果 一手烟组,三手烟组与对照组相比,EPCs百分比显著下降[分别 (0.045±0.029)% vs (0.099±0.023) %,(0.047±0.023)% vs (0.099±0.023) %,均P<0.001),MPO, IL-6,TNF-α水平显著升高[分别为MPO: 1.39±0.26) U/g vs (0.48±0.17) U/g,(1.35±0.32) U/g vs (0.48±0.17) U/g;IL-6:(3.83±0.13) ng/ml vs (0.79±0.12) ng/ml,(3.45±0.15) ng/ml vs (0.79±0.12) ng/ml;TNF-α:(1.77±0.26) ng/ml vs (0.81±0.21) ng/ml,(1.78±0.23) ng/ml vs (0.81±0.21) ng/ml, 均P<0.001),NO水平显著降低[分别为(0.057±0.0063) μmol/L vs (0.080±0.0067) μmol/L,(0.060±0.0072) μmol/L vs (0.080±0.0067) μmol/L,均P<0.001);一手烟组,三手烟组比较各指标差异无统计学意义(均P>0.05),光镜下可见观察一手烟组,三手烟组与对照组相比心肌组织发生明显损伤.结论 三手烟可通过氧化应激和炎症反应降低小鼠内皮祖细胞水平,损伤小鼠心肌组织,作用程度与一手烟相似.     

Determination of plasma lead levels in normal subjects and in lead-exposed workers

ABSTRACT Lead levels in whole blood and in plasma were measured in 64 non-exposed and in 29 exposed subjects with signs and symptoms of varying severity. Lead was determined by atomic absorption spectrophotometry after chelation with ammonium pyrrolidine dithiocarbamate and extraction with methyl isobutyl ketone. The method has a sensitivity of 0·4 μg/100 ml (0·02 μmol/l) for whole blood and of 0·2 μg/100 ml (0·01 μmol/l) for plasma and is reliably accurate and precise. Plasma lead increases progressively and significantly with the increase of whole blood lead, while its relative percentage in the plasma remains practically constant at all concentrations in whole blood. In exposed subjects a highly significant correlation was found between lead in plasma and lead in urine (r = 0·549) but the correlation coefficient was higher for whole blood lead versus urinary lead (r = 0·938). Aminolevulinic acid excretion in urine appeared to be significantly related to plasma lead concentration (r = 0·563) but to a greater extent to whole blood levels (r = 0·801). There was no significant correlation between lead in plasma and the logarithm of aminolevulinic acid dehydratase. The hypothesis is advanced that plasma lead, the more biologically active fraction of the metal, could be related to different individual sensitivities which would condition the development of toxic effects in various organs at different levels of lead.     

A simple colorimetric method for the determination of primaquine metabolites in urine

A simple method of screening large numbers of urine samples for the presence of primaquine metabolites has been developed using a commercially available diazonium salt reagent and an extraction cartridge. The extraction requires only 1.0 ml of urine and is selective for acidic or neutral primaquine metabolites. With primaquine-dosed rats, primaquine metabolites could be detected 36 hours after administration of the drug. The sensitivity of the method was found to be approximately 400 μg/l.     

Arsenic species excretion in a group of persons in northern Germany--contribution to the evaluation of reference values

Reference values for four arsenic species (inorganic As(III); inorganic As(V); dimethylarsinic acid DMA; monomethylarsonic acid MMA) in urine were evaluated for 101 male persons in northern Germany (46.9 ± 10.5 y) applying anion exchange chromatographic species separation with on-line hydride-technique atomic absorption spectrometry (between-days imprecision 6.8-10.1 %; 11.0-50.0 μg/l; n = 30; detection limits d. l. 1; 10; 2; 2 μg/l). DMA was found in 88.1 % of the persons (x ± s = 7.21 ± 9.64 μg/g creatinine; median 4.02 μg/g; 95 % < 22.5 μg/g) followed by As(III) (11.3 %; median < d. l.; 95 % < 0.54 μg/g) whereas no MMA and As(V) could be found. Seafood consumption within the last 2 days led to higher DMA levels compared to having seafood more than 6 days ago (n = 43 vs. n = 42; 10.04 ± 11.58 vs. 3.47 ± 3.55 μg/g; p < 0.01).     

Antimicrobial susceptibility of gonococci isolated in the Central African Republic

Using agar dilution techniques, we determined the minimum inhibitory concentrations (MIC) of 11 antimicrobials for 70 isolates of Neisseria gonorrhoeae obtained in Bangui, Central African Republic. These gonococci were found to be fairly susceptible to commonly used antibiotics: only 3 isolates (4%) had a penicillin MIC ≥ 1.0 μg/ml and 6 (9%) had a tetracycline MIC ≥ 2.0 μg/ml. With regard to other antibiotics, 54 isolates (77%) had an erythromycin MIC ≥ 0.25 μg/ml, all had a spectinomycin MIC ≤ 16 μg/ml, and 32 (46%) had a sulfamethoxazole/trimethoprim MIC ≥ 9.5/0.5 μg/ml. None of these isolates produced penicillinase.     

A Rapid Method for the Determination of Delta Amino-laevulinic Acid in Urine

A rapid colorimetric method, sensitive to 0·5 μg., is described for the determination of delta amino-laevulinic acid in urine. The method consists of a modification of the picrate methods of Shuster in which interference by porphobilinogen is avoided by the use of an ion exchange resin. The method is found to give results which compare in accuracy with the longer standard method of Mauzerall and Granick.     

Blood lead and the symptoms of lead absorption

ABSTRACT Eighty-one percent of all hourly paid men who had been employed for more than six months in a factory making lead acid batteries and plastics completed a modified Cornell medical index health questionnaire. Blood lead and erythrocyte protoporphyrin (EPP) were also measured. The questions were grouped into symptom categories as follows: all physical, all psychological, “potentially lead induced,” pulmonary, cardiovascular, gastrointestinal, skin, nervous system, genitourinary, and fatigue. For each symptom category the pooled percentages of men whose symptom scores were above the common median of the three blood lead groups 10-, 40-, and 60 and over μg/100 ml (0·48-, 1·93-, and 2·90 and over μmol/l) within age/smoking subgroups were calculated. In every symptom category the percentages in the two lower blood lead groups differed little, but the percentages were consistently higher in men with blood concentration of 60 μg/100 ml (2·90 μmol/l) and over. Differences between a combined 10-59 μg/100 ml (0·48-2·85 μmol/l) blood lead group and the 60 and over μg/100 ml (≥2·90 μmol/l) group were statistically significant at the 0·01 level for “potentially lead induced” symptoms and at the 0·05 level for skin and psychological symptoms. Broadly similar results were obtained with four log₁₀ EPP groups 0·6-, 1·5-, 1·7-, and ≥2·0, but differences did not reach statistical significance. There was no obvious explanation as to why symptoms that are not found in classic lead poisoning should be increased almost as much as those that are. It was thought that these results could be biased due to the men's knowledge of the symptoms associated with lead exposure, but the possibility that they may be partly due to lead absorption cannot be excluded.     

Chloroquine and desethylchloroquine concentrations during regular long-term malaria prophylaxis

The concentrations of chloroquine and desethylchloroquine in the blood of 10 healthy adult Swedish volunteers who had been taking 310 mg chloroquine base once a week for at least 8 months for malaria prophylaxis were measured. Samples of capillary whole blood from the volunteers were dried on filter-paper and the drug and its principal metabolite determined by a specific high-performance liquid chromatography (HPLC) method. The day after taking the drug, the mean concentration of chloroquine and desethylchloroquine in whole blood were 1305 nmol/l and 915 nmol/l, respectively, and immediately before the next weekly dose, 489 nmol/l and 384 nmol/l, respectively. These are considered to be greater than the minimum inhibitory concentrations for susceptible strains but less than the maximum tolerated concentrations. The dosage of chloroquine recommended roughly 40 years ago for regular long-term prophylaxis should therefore not be changed.     

An investigation of modifying effects of metallothionein single-nucleotide polymorphisms on the association between mercury exposure and biomarker levels

Background: Recent studies have suggested that several genes that mediate mercury metabolism are polymorphic in humans.Objective: We hypothesized that single-nucleotide polymorphisms (SNPs) in metallothionein (MT) genes may underlie interindividual differences in mercury biomarker levels. We studied the potential modifying effects of MT SNPs on mercury exposure–biomarker relationships.Methods: We measured total mercury in urine and hair samples of 515 dental professionals. We also surveyed occupational and personal exposures to dental amalgam and dietary fish consumption, from which daily methylmercury (MeHg) intake was estimated. Log-transformed urine and hair levels were modeled in multivariable linear regression separately against respective exposure surrogates, and the effect modification of 13 MT SNPs on exposure was investigated.Results: The mean mercury levels in urine (1.06 μg/L) and hair (0.51 μg/g) were not significantly different from the U.S. general population (0.95 μg/L and 0.47 μg/g, respectively). The mean estimated daily MeHg intake was 0.084 μg/kg/day (range, 0–0.98 μg/kg/day), with 25% of study population intakes exceeding the current U.S. Environmental Protection Agency reference dose of 0.1 μg/kg/day. Multivariate regression analysis showed that subjects with the MT1M (rs2270837) AA genotype (n = 10) or the MT2A (rs10636) CC genotype (n = 42) had lower urinary mercury levels than did those with the MT1M or MT2A GG genotype (n = 329 and 251, respectively) after controlling for exposure and potential confounders. After controlling for MeHg intake, subjects with MT1A (rs8052394) GA and GG genotypes (n = 24) or the MT1M (rs9936741) TT genotype (n = 459) had lower hair mercury levels than did subjects with MT1A AA (n = 113) or MT1M TC and CC genotypes (n = 15), respectively.Conclusion: Our findings suggest that some MT genetic polymorphisms may influence mercury biomarker concentrations at levels of exposure relevant to the general population.     

Determination of Lead in Urine by Atomic Absorption Spectrophotometry

A method for the determination of lead in urine by means of atomic absorption spectrophotometry (AAS) is described. A combination of wet ashing and extraction with ammonium pyrrolidine dithiocarbamate into isobutylmethylketone was used. The sensitivity was about 0·02 μg./ml. for 1% absorption, and the detection limit was about 0·02 μg./ml. with an instrumental setting convenient for routine analyses of urines. Using the scale expansion technique, the detection limit was below 0·01 μg./ml., but it was found easier to determine urinary lead concentrations below 0·05 μg./ml. by concentrating the lead in the organic solvent by increasing the volume of urine or decreasing that of the solvent. The method was applied to fresh urines, stored urines, and to urines, obtained during treatment with chelating agents, of patients with lead poisoning. Urines with added inorganic lead were not used. The results agreed well with those obtained with a colorimetric dithizone extraction method (r = 0·989). The AAS method is somewhat more simple and allows the determination of smaller lead concentrations.     

Association of renal function and delta-aminolevulinic acid dehydratase polymorphism among Vietnamese and Singapore workers exposed to inorganic lead

Objectives

To investigate the effect of δ‐aminolevulinic acid dehydratase (ALAD) polymorphisms on the association between blood lead and renal function among Vietnamese and Singaporean workers who were exposed to low to medium levels of inorganic lead, and to study the distribution of ALAD polymorphism among Vietnamese, Chinese, Malays and Indians.

Methods

A total of 459 male and female workers were studied. Blood and urine were collected for each worker in order to determine ALAD genotype, blood lead, and urinary δ‐aminolevulinic acid (ALAU). Renal function tests included urine albumin (Ualb), urine β2 microglobulin (Uβ2m), urinary α1 microglobulin (Uα1m), N‐acetyl‐glucosaminidas (NAG), and urine retinol blinding protein (RBP). A multiple regression model with interaction term was applied to fit the entire data and to explore the modifying effect of ALAD polymorphism on the relation of blood lead to each renal function parameter.

Results

ALAD1‐1 was the predominant genotype for all the ethnic groups while ALAD2‐2 was the rarest. The frequency of ALAD2 allele was higher among Malays (8.8%) and Indians (10.6%) compared to the Chinese (5.0%) and Vietnamese (4.3%). The geometric mean of blood lead for all workers was 19.0 μg/dl. The models for Uβ2m, Uα1m, and NAG showed that the ALAD1‐2/2‐2 group had higher β coefficients than the ALAD1‐1 group. Corresponding to 10 μg/dl blood lead, ALAD1‐1 homozygotes had an increment of 1.288 μg/g Cr, 1.175 mg/g Cr, and 1.995 U/g Cr for Uβ2m, Uα1m, and NAG, respectively. ALAD1‐2/2‐2 subjects had higher increments of 3.802 μg/g Cr, 2.138 mg/g Cr, and 3.89 U/g Cr for Uβ2m, Uα1m, and NAG, respectively.

Conclusion

The frequency of the ALAD2 allele is as low in Vietnamese workers as in Chinese. Workers with the ALAD2 allele appeared more susceptible to the effects of lead (especially at higher levels) on renal function.     

Population-based inorganic mercury biomonitoring and the identification of skin care products as a source of exposure in New York City

Background

Mercury is a toxic metal that has been used for centuries as a constituent of medicines and other items.

Objective

We assessed exposure to inorganic mercury in the adult population of New York City (NYC).

Methods

We measured mercury concentrations in spot urine specimens from a representative sample of 1,840 adult New Yorkers in the 2004 NYC Health and Nutrition Examination Survey. Cases with urine concentrations ≥ 20 μg/L were followed up with a telephone or in-person interview that asked about potential sources of exposure, including ritualistic/cultural practices, skin care products, mercury spills, herbal medicine products, and fish.

Results

Geometric mean urine mercury concentration in NYC was higher for Caribbean-born blacks [1.39 μg/L; 95% confidence interval (CI), 1.14–1.70] and Dominicans (1.04 μg/L; 95% CI, 0.82–1.33) than for non-Hispanic whites (0.67 μg/L; 95% CI, 0.60–0.75) or other racial/ethnic groups. It was also higher among those who reported at least 20 fish meals in the past 30 days (1.02 μg/L; 95% CI, 0.83–1.25) than among those who reported no fish meals (0.50 μg/L; 95% CI, 0.41–0.61). We observed the highest 95th percentile of exposure (21.18 μg/L; 95% CI, 7.25–51.29) among Dominican women. Mercury-containing skin-lightening creams were a source of exposure among those most highly exposed, and we subsequently identified 12 imported products containing illegal levels of mercury in NYC stores.

Conclusion

Population-based biomonitoring identified a previously unrecognized source of exposure to inorganic mercury among NYC residents. In response, the NYC Health Department embargoed products and notified store owners and the public that skin-lightening creams and other skin care products that contain mercury are dangerous and illegal. Although exposure to inorganic mercury is not a widespread problem in NYC, users of these products may be at risk of health effects from exposure.     

The Aral Sea disaster--human biomonitoring of Hg, As, HCB, DDE, and PCBs in children living in Aralsk-and Akchi, Kazakhstan

Mercury and arsenic have been measured in urine samples and HCB, DDE and PCBs in blood samples of children from Aralsk and Akchi, Kazakhstan. Due to the special situation of Aralsk in the desert left by the drying out Aral Sea, environmental pollution with heavy metals and organic contaminants is believed to be higher than elsewhere in Kazakhstan. Aralsk was formerly located at the shore of the Aral Sea and is now far away from it. Akchi is a similar village and was included in this study as a Kazakh reference site. Urine concentrations of arsenic were higher in Akchi (9.4 μg/l) than in Aralsk (5.5 μg/l) and compared to children from Mannheim, Germany (4.25 μg/l; Median values). Regarding Hg, differences between children of Aralsk and Akchi were not significant and concentrations were lower than reference values from Germany. DDE contamination of children from Aralsk (2.48 μg/l) was significantly higher compared to Akchi (1.35 μg/l). DDE concentrations in blood samples from children in both cities were also significantly higher than the German reference value (0.7 μg/l). HCB and PCBs levels differed significantly between both Kazakh groups. However, concentrations of these compounds were lower than German reference values and there was no significant difference to samples from Mannheim children.     

Biomarkers of chlorpyrifos exposure and effect in Egyptian cotton field workers

Background

Chlorpyrifos (CPF), a widely used organophosphorus pesticide (OP), is metabolized to CPF-oxon, a potent cholinesterase (ChE) inhibitor, and trichloro-2-pyridinol (TCPy). Urinary TCPy is often used as a biomarker for CPF exposure, whereas blood ChE activity is considered an indicator of CPF toxicity. However, whether these biomarkers are dose related has not been studied extensively in populations with repeated daily OP exposures.

Objective

We sought to determine the relationship between blood ChE and urinary TCPy during repeated occupational exposures to CPF.

Methods

Daily urine samples and weekly blood samples were collected from pesticide workers (n = 38) in Menoufia Governorate, Egypt, before, during, and after 9–17 consecutive days of CPF application to cotton fields. We compared blood butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities with the respective urinary TCPy concentrations in each worker.

Results

Average TCPy levels during the middle of a 1- to 2-week CPF application period were significantly higher in pesticide applicators (6,437 μg/g creatinine) than in technicians (184 μg/g) and engineers (157 μg/g), both of whom are involved in supervising the application process. We observed a statistically significant inverse correlation between urinary TCPy and blood BuChE and AChE activities. The no-effect level (or inflection point) of the exposure–effect relationships has an average urinary TCPy level of 114 μg/g creatinine for BuChE and 3,161 μg/g creatinine for AChE.

Conclusions

Our findings demonstrate a dose–effect relationship between urinary TCPy and both plasma BuChE and red blood cell AChE in humans exposed occupationally to CPF. These findings will contribute to future risk assessment efforts for CPF exposure.     

Cadmium metabolism in man

ABSTRACT Twenty-one high frequency solderers, who had been exposed to cadmium (Cd) from a solder for periods ranging from 1 month to 18 years (median 8 months; present time-weighted average 30 nmol/m³; particle size below 1μm) had Cd levels ranging from < 10 to 440 nmol/l in blood and from < 0·5 to 27 μmol/mol creatinine in urine. Individual workers showed considerable variations in blood Cd levels with time, but less variation in urine levels. There was a statistically significant (p < 0·001) increase of Cd in urine with increasing exposure time. Four gas solderers, who had been intermittently exposed for 8-20 years (median 17 years) had Cd levels ranging from 45 to 150 nmol/l and urine levels of from 2 to 20 μmol/mol creatinine. There was no correlation between Cd levels in blood and urine during exposure. After exposure had ceased there was a considerable decay of blood Cd in most subjects. The half-time in 11 people ranged from 25 to 146 days (median 41 days). After the decay blood levels reached a steady state. Concentrations in urine did not decrease, or did so only very slowly. There was a significant increase of levels in urine (p < 0·001) with increasing post-decay levels in blood. There was also a significant increase (0·01 < p < 0·05) of excretion of ß₂-microglobulin in urine (range 1·1-18 mg/mol creatinine, median 4·7 mg/mol creatinine) measured 11-15 months after exposure had ceased, with increasing Cd levels in urine. This may indicate an effect on renal tubular function even at kidney Cd loads corresponding to Cd levels in urine of the order of as little as 10 μmol/mol creatinine.