Novel urinary biomarkers and their association with urinary heavy metals in chronic kidney disease of unknown aetiology in Sri Lanka: a pilot study

Introduction: Chronic kidney disease of unknown etiology (CKDu) has emerged as a significant public health problem in Sri Lanka. The role of environmental exposure to cadmium and arsenic in the aetiology of CKDu is still unclear. Identification of a panel of novel urinary biomarkers would be invaluable in the study of toxin mediated damage postulated to be the aetiology of CKDu. Objectives: The aims of this study were to evaluate the profile of novel urinary biomarkers in CKDu patients and identify any association with environmental exposure to heavy metals. Methods: Thirty seven randomly selected CKDu patients attending a renal clinic in the North Central Province and two control groups namely a farmer group (n=39) and a non-farmer group (n=40) from a non-endemic area were included in this comparative cross sectional study. Urine samples were analyzed for heavy metals and five urinary biomarkers. Results: CKDu patients had significantly elevated urinary levels of fibrinogen (198.2 ng/mg creatinine p<0.001), clusterin (3479 ng/mg creatinine p<0.001), cystatin-C (5124.8 ng/mg creatinine p<0.001) and ß2-microglobulin (9913.4 ng/mg creatinine p<0.001) compared to the control groups. Fibrinogen and ß2-microglobulin were the best to discriminate CKDu patients from normal individuals with the receiver operator areas under the curve being 0.867 and 0.853, respectively. Urinary fibrinogen and KIM-1 levels correlated positively with urinary arsenic levels. KIM-1 levels correlated positively with urinary mercury and lead levels but no correlation was seen with urinary cadmium levels. Conclusions: Fibrinogen and ß2-microglobulin have the potential of being a screening tool for detection of CKDu and may aid the early diagnosis of toxin mediated tubular injury in CKDu. Their usefulness need to be further validated in a larger epidemiological study of patients with early stages of CKDu.

The independent contribution of bone and erythrocyte lead to urinary lead among middle-aged and elderly men: the normative aging study.

Plasma is the component of blood from which lead is free to cross cell membranes and cause organ toxicity. Plasma lead levels, however, are extremely low and difficult to measure. Urinary lead originates from plasma lead that has been filtered at the glomerular level; thus, urinary lead adjusted for glomerular filtration rate serves as a proxy for plasma lead levels. In this investigation we examined the interrelationships of lead levels in whole blood corrected by hematocrit [i.e., erythrocyte lead (EPb)], trabecular bone (TBoPb), cortical bone (CBoPb), and urine excreted over 24 hr (UPb); all samples were obtained from 71 middle-aged and elderly men with no known occupational lead exposures. Lead was measured by graphite furnace atomic absorption spectroscopy (blood), K-X-ray fluorescence (bone), and inductively coupled plasma mass spectroscopy (urine). Lead levels were generally low, with mean EPb, TBoPb, and CBoPb values of 13.8, 31.1, and 21.7 microg/g, respectively, and a median UPb value of 6.15 microg/day. In generalized additive models adjusted for body weight and creatinine clearance rate, both EPb and bone lead variables remained independently and significantly associated with UPb. This finding suggests that bone influences plasma lead in a manner that is independent of the influence of erythrocytic lead on plasma lead. Thus, the superiority of bone lead over blood lead in predicting some chronic forms of toxicity may be mediated through bone's influence on plasma lead. In addition, this study suggests that measurement of urinary lead might be useful as a proxy for plasma lead levels in studies of lead toxicity.

Influence of bone resorption on the mobilization of lead from bone among middle-aged and elderly men: the Normative Aging Study.

Bone stores of lead accrued from environmental exposures and found in most of the general population have recently been linked to the development of hypertension, cognitive decrements, and adverse reproductive outcomes. The skeleton is the major endogenous source of lead in circulating blood, particularly under conditions of accelerated bone turnover and mineral loss, such as during pregnancy and in postmenopausal osteoporosis. We studied the influence of bone resorption rate on the release of lead from bone in 333 men, predominantly white, middle-aged and elderly (mostly retired) from the Boston area. We evaluated bone resorption by measuring cross-linked N-telopeptides of type I collagen (NTx) in 24-hr urine samples with an enzyme-linked immunosorbent assay. We used K-X-ray fluorescence to measure lead content in cortical (tibia) and trabecular (patella) bone; we used graphite furnace atomic absorption spectroscopy and inductively coupled plasma mass spectroscopy to measure lead in blood and urine, respectively. After adjustment for age and creatinine clearance, the positive relation of patella lead to urinary lead was stronger among subjects in the upper two NTx tertiles (beta for patella lead > or =0.015) than in the lowest NTx tertile (beta for patella lead = 0.008; overall p-value for interactions = 0.06). In contrast, we found no statistically significant influence of NTx tertile on the relationship of blood lead to urinary lead. As expected, the magnitude of the relationship of bone lead to urinary lead diminished after adjustment for blood lead. Nevertheless, the pattern of the relationships of bone lead to urinary lead across NTx tertiles remained unchanged. Furthermore, after adjustment for age, the relation of patella lead to blood lead was significantly stronger in the upper two NTx tertiles (beta for patella lead > or =0.125) than in the lowest NTx tertile (beta for patella lead = 0.072). The results provide evidence that bone resorption influences the release of bone lead stores (particularly patella lead) into the circulation.

Validation of K x-ray fluorescence bone lead measurements by inductively coupled plasma mass spectrometry in cadaver legs.

K x-ray fluorescence (KXRF) systems are being used in a growing number of epidemiologic studies to measure bone lead levels as a biological marker of accumulated lead exposure. Although validation of the KXRF technique in lead-doped phantoms and bare bone specimens has been repeatedly demonstrated, few studies have compared KXRF to chemical measurements of actual intact cadaver limbs (with skin and soft tissues). In this study, lead levels in eight amputated human legs were measured by KXRF; after dissection, levels in the bare bones were again measured by KXRF and then by inductively coupled plasma mass spectrometry (ICP-MS). We observed close agreement between the KXRF and ICP-MS measurements with correlation coefficients for both the tibia and patella greater than 0.9. In this study we provide further support for the validity of KXRF measurements, particularly with respect to the patella.

K x-ray fluorescence measurements of bone lead concentration: the analysis of low-level data.

K line x-ray fluorescence (KXRF) measurements of bone lead have emerged as a promising new biological marker of internal lead dose in epidemiological studies. Some disagreements exist, however, over the analysis of data at low levels of bone lead concentration. In this study, we performed 30 serial measurements on each of three phantoms containing spiked amounts of lead. Chemical analysis of these phantoms using an inductively coupled plasma mass spectrometer (ICPMS) indicated that the lead concentrations were 0.30, 5.77, and 11.57 micrograms g-1. Analysis of the data was performed using several definitions of a minimum detectable limit (MDL) to recode data below the MDL, and using all of the continuous point estimates of lead concentration in the phantom (including negative estimates). The results demonstrate that the use of MDLs to recode low-level observations reduces the efficiency of the analysis and the ability to distinguish between the phantoms. Retaining all point estimates of KXRF-measured bone lead concentration provides less bias and greater efficiency in comparing the mean or median levels of bone lead of different populations.

Improvements in the calibration of 109Cd K x-ray fluorescence systems for measuring bone lead in vivo.

A 109Cd K x-ray fluorescence (XRF) system using a point source in a back-scatter geometry is described. The suitability of plaster-of-Paris phantoms as targets for intercalibration standards was evaluated. When the phantom concentrations were measured by inductively-coupled-plasma mass spectrometry (ICPMs), the calculated phantom concentrations underestimated true concentrations by an average of 15%. Since calculated values are used to calibrate the K XRF system, in vivo bone-lead concentrations may be similarly underestimated. The difference between calculated and measured concentration is attributable to impurities in the plaster of Paris (e.g. calcium carbonate). The ICPMS-measured concentrations were used to calibrate the K XRF system. The same phantoms were also measured as 'unknowns' by a bone-lead measurement system (Abiomed, Danvers, MA, USA). The commercial system overestimated the lowest-concentration phantoms and underestimated the phantoms with concentrations above 15 microg Pb/g plaster of Paris. The commercial system and our system were compared by measurement of the new phantoms in air and in water. The K XRF system exhibited better precision in both situations. On the basis of this work, we recommend that plaster-of-Paris phantoms used to calibrate K XRF measurement systems be analysed first by ICPMS or another valid analytical technique.

Environmental lead contamination and pediatric lead intoxication in an Andean Ecuadorian village.

Environmental lead (Pb) contamination was measured in samples of soil and locally grown food produce in a remote Ecuadorian village where Pb glazing of ceramics is the local cottage industry. The Pb concentration levels of local soil samples collected at varying distances from a cluster of backyard Pb baking kilns were 29,213 ppm (microg/g) at 0.001 km, 172 ppm at 0.005 km, 81 ppm at 0.01 km, 55 ppm at 1 km, 19 ppm at 2 km, and 1.4 ppm at 6 km, significantly higher than levels in control soil samples from non-Pb-glazing reference areas. Samples of locally grown food produce were also found to be Pb contaminated. Venous blood samples from 166 schoolchildren (ages 4 months to 15 years) in the study area and 56 children in the reference area showed mean blood lead levels of 40.0 microg/dl (SD: 24.5; range: 6.2. - 119.1 microg/dL) and 6.6 microg/dL (SD: 3.4; range: 1.9 - 18.1 microg/dL), respectively, which were significantly different (p = 0.0001). The Pb levels in milk from breastfeeding mothers ranged from 1.44 to 39 ng/g. Lead isotope ratios of the children's blood and of samples of village soil revealed a common Pb source or "fingerprint."

Determination of total arsenic concentrations in nails by inductively coupled plasma mass spectrometry.

The analysis of trace elements in biological samples will extend our understanding of the impact that environmental exposure to these elements has on human health. Measuring arsenic content in nails has proven useful in studies evaluating the chronic body burden of arsenic. In this study, we developed methodology with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of total arsenic in nails. We assessed the utility of the washing procedures for removing surface contamination. Four types of preanalysis treatments (water bath, sonication, water bath plus sonication, and control) after sample decomposition by nitric acid were compared to evaluate the digestion efficiencies. In addition, we studied the stability of the solution over 1 wk and the effect of acidity on the arsenic signal. Arsenic content in the digested solution was analyzed by using Ar-N2 plasma with Te as the internal standard. The results suggest that washing once with 1% Triton X-100 for 20 min for cleaning nail samples prior to ICP-MS analysis is satisfactory. Repeated measurement analysis of variance revealed that there was no significant difference among the various sample preparation techniques. Moreover, the measurements were reproducible within 1 wk, and acidity seemed to have no substantial influence on the arsenic signal. A limit of detection (on the basis of three times the standard deviation of the blank measurement) of 7 ng As/g toenail was achieved with this system, and arsenic recoveries from reference materials (human hair and nails) were in good agreement (95-106% recovery) with the certified/reference values of the standard reference materials. ICP-MS offers high accuracy and precision, as well as high-throughput capacity in the analysis of total arsenic in nail samples.

Pectin with low molecular weight and high degree of esterification increases absorption of 58Fe in growing rats.

Effects of pectins with different degrees of esterification (DE) and molecular weights (MW) on iron bioavailability were investigated in healthy growing rats by following erythrocyte incorporation of a dose of 58Fe. Rats were fed a control diet for 8 d and then deprived of food for 16 h. Two hours after the start of feeding iron-deficient diets, with or without pectin (80 g/kg diet), a dose of FeSO4 rich in 58Fe (60.28%) was intubated into the stomach; rats were then allowed to feed for an additional 4 h before withdrawal of food for 10 h. Rats were then fed iron-adequate diets for 9 d. The pectins differed in DE and MW, respectively, as follows: P-A (73%, 860,000), P-B (75%, 89,000), P-C (22%, 1,260,000) and P-D (24%, 114,000). Rats fed pectin-free diet with free access to food or restricted to the same quantity consumed by a respective pectin group served as controls. Iron absorption was 48% in the control group and 57% in rats fed P-B. Rats fed P-B had higher (P 2 < or = 0.05) serum iron, transferrin saturation, hematocrit and liver and spleen iron than the control group or the group fed P-C. These indices, except for transferrin saturation, were also higher In rats fed P-A and P-D compared with those fed P-C and controls, but to a lesser extent than in rats fed P-B. The data indicate that bioavailability of dietary non-heme iron was enhanced when pectin of low MW and high DE was added to the diet. This improvement was not evident with pectins having high MW and/or low DE.

Determination of the total arsenic concentration in human urine by inductively coupled plasma mass spectrometry: a comparison of the accuracy of three analytical methods.

Measurement of arsenic (As) in biological samples such as urine has important clinical applications and is being undertaken more frequently in epidemiologic studies because of concern about the carcinogenicity of low to moderate levels of As exposure. The objective of this study was to evaluate and improve the accuracy of As determination in urine by inductively coupled plasma mass spectrometry (ICP-MS). Determination of As in biological samples by ICP-MS is difficult for two reasons: the formation of the molecular ion 40Ar35Cl, which overlaps with monoisotopic As at a mass-to-charge ratio (m/z) of 75 (causing spectral interference), and signal enhancement due to organic matrix (nonspectral interference). Available procedures were examined, including the application of different correction procedures using 40Ar37Cl and 16O35Cl molecular-ion formation; the addition of N2 into plasma or nebulizer gas flows; and the addition of organic molecules to the sample and to calibration standards to eliminate or correct for interference due to molecular-ion formation. The accuracy and precision of determination of As [m/z 75, ionization potential (IP) 9.81 eV] with use of an internal standard was also investigated. Three elements were studied as candidate internal standards: germanium (Ge: m/z 74, IP 7.90 eV), indium (In: m/z 115, IP 5.79 eV), and tellurium (Te: m/z 128, IP 9.01 eV). It was found that these three elements performed more or less equally well with Ar-N2 plasma; it was also found that accuracy was significantly improved when Te was used as the internal standard instead of Ge or In for ethanol-added samples. Our results indicate that accurate and precise measurement of As in urine by ICP-MS can be obtained by either of two methods (< 5% error, approximately 2% RSD, limit of detection 0.1 ng ml-1): (1) the addition of 1% N2 to plasma gas flow or 3% N2 to nebulizer gas flow, along with use of any of the internal standards tested, or (2) the addition of ethanol to the sample and to calibration standards, with use of Te as the internal standard. The most accurate results (< 1% error) for National Institute of Standard and Technology Standard Reference Material (NIST SRM) 2670 (toxic elements in urine) were obtained with Ar-N2 plasma with either Te or In as the internal standard.

Effect of iron deficiency anemia on lead distribution after intravenous dosing in rats.

OBJECTIVE: To determine the effect of iron deficiency anemia on blood and tissue lead distribution. METHODS: 24 weanling rats were divided into 2 groups. One group received an iron replete diet (200 ppm); the other received a low iron diet (20 ppm). After 24 days, each group was further subdivided into two doses of lead (5 mg/kg and 10 mg/kg) which was administered intravenously. Rats were continued on their respective diets for 7 days post-lead injection to allow tissue distribution, then sacrificed and blood and tissue lead concentration measured. RESULTS: Prior to lead administration, baseline blood lead concentrations were not significantly different between groups. At sacrifice, whole blood lead levels were significantly higher in iron deficient animals than in iron replete at both 5 and 10 mg/kg administered lead. Iron deficient animals had comparable lead concentrations to iron replete animals in brain, kidney and liver. Femur lead concentrations were higher at 10 mg/kg administered lead. CONCLUSION: Iron deficiency alters lead distribution such as that increased lead is found in blood for a given exposure.