Sidney M. Hopfer

Nickel absorption and kinetics in human volunteers

Abstract Mathematical modeling of the kinetics of nickel absorption, distribution, and elimination was performed in healthy human volunteers who ingested NiSO4 drinking water (Experiment 1) or added to food (Experiment 2). Nickel was analyzed by electrothermal atomic absorption spectrophotometry in serum, urine, and feces collected during 2 days before and 4 days after a specified NiSO4 dose (12 μg of nickel/kg, n = 4; 18 μg of nickel/kg, n = 4; or 50 μg of nickel/kg, n = 1). In Experiment 1, each of the subjects fasted 12 hr before and 3 hr after drinking one of the specified NiSO4 doses dissolved in water; in Experiment 2, the respective subjects fasted 12 hr before consuming a standard American breakfast that contained the identical dose of NiSO4 added to scrambled eggs. Kinetic analyses, using a compartmental model, provided excellent goodness-of-fit for paired data sets from all subjects. Absorbed nickel averaged 27 ± 17% (mean ± SD) of the dose ingested in water vs 0.7 ± 0.4% of the same dose ingested in food (a 40-fold difference); rate constants for nickel absorption, transfer, and elimination were not significantly influenced by the oral vehicle. The elimination half-time for absorbed nickel averaged 28 ± 9 hr. Renal clearance of nickel averaged 8.3 ± 2.0 ml/min/1.73 m2 in Experiment 1 and 5.8 ± 4.3 ml/min/1.73 m2 in Experiment 2. This study confirms that dietary constituents profoundly reduce the bioavailability of Ni2+ for alimentary absorption; approximately one-quarter of nickel ingested in drinking water after an over-night fast is absorbed from the human intestine and excreted in urine, compared with only 1% of nickel ingested in food. The compartmental model and kinetic parameters provided by this study will reduce the uncertainty of toxicologic risk assessments of human exposures to nickel in drinking water and food.

Detection of two Zn-finger proteins of Xenopus laevis, TFIIIA, and p43, by probing western blots of ovary cytosol with 65Zn2+, 63Ni2+, or 109Cd2+.

Tow Zn-finger proteins, TFIIIA (a constituent of 7S RNP particles) and p43 (a constituent of 42S RNP particles), were detected in ovary extracts of juvenileXenopus laevis females by in vitro binding of radiolabeled divalent metals. Proteins fractionated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) were transferred by Western blotting onto nitrocellulose membranes, probed with65Zn2+,63Ni2+, or109Cd2+, and visualized by autoradiography. Detection limits for TFIIIA were approx 0.07 μg/well by109Cd2+-probing, 0.13 μg/well by65Zn2+-probing, and 0.26 μ/well by63Ni2+-probing. Protein p43 was more clearly visualized by probing with63Ni2+ than with65Zn2+ or109Cd2+. After purified TFIIIA was cleaved with cyanogen bromide,65Zn2+,109Cd2+, and63Ni2+ distinctly labeled the 22 kDa middle fragment;65Zn2+ and109Cd2+ also labeled the 11 kDa N-terminal fragment, but didnot label the 13 kDa C-terminal fragment. These results are consistent with the notion that the radioligands were bound to finger-loop domains of TFIIIA, which occur in the middle and N-terminal fragments. Based on the abilities of nonradioactive metal ions to compete with65Zn2+ for binding to TFIIIA on Western blots, the relative affinities of the metals for TFIIIA were ranked as follows: Zn2+=Cu2+≥Hg2+>Cd2+>Co2+≥Ni2+. Even at a 1000-fold molar excess, Mn2+ did not compete with65Zn2+ for binding to TFIIIA. Probing Western blots with the radiolabeled metal ions greatly facilitates the detection, isolation, and quantitation of TFIIIA and p43.

Teratogenicity of Ni2+ inXenopus laevis, assayed by the FETAX procedure

The teratogenicity of Ni2+ was tested by the FETAX (Frog Embryo Teratogenesis Assay:Xenopus) procedure in the South African frog,Xenopus laevis. In seven assays, beginning at 5 h postfertilization, groups ofXenopus embryos were incubated for 96 h in media that contained Ni2+ (added as NiCl2) at concentrations ranging from 1 × 10−7 to 3 × 10−3 mol/L; control groups were incubated in the same medium without added NiCl2. At 101 h postfertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to determine their developmental stages, malformations, and head-to-tail lengths. In control embryos, survival was ≥95% and malformations were ≤7%. Malformations were found in >95% of embryos exposed to Ni2+ concentrations ≥5.6 μmol/L. The most frequent malformations in Ni2+-exposed embryos were ocular, skele|tal, and intestinal deformities; less common malformations included facial, cardiac, and integumentary deformities. Other abnormalities, not categorized as malformations, included stunted growth, dermal hypopigmentation, and coelomic effusions or hemorrhages. Themedian embryolethal concentration (LC50) of Ni2+ was 365 (SE±9) μmol/L; themedian teratogenic concentration (EC50) was 2.5 (SE±0.1) μmol/L; theTeratogenic Index (TI=LC50/EC50) was 147 (SE±5), indicating that Ni2+-exposures were limited to specific 24 h periods showed thatXenopus embryos were most susceptible to Ni2+-induced malformations on the second and third days of life, during the most active period of organogenesis.

Intraarticular carcinogenesis bioassays of CoCrMo and TiAlV alloys in rats

Wear-debris powders of cobalt-chromium-molybdenum (CoCrMo) and titanium-aluminum-vanadium (TiAlV) alloys, which are widely used for orthopedic implants (eg, hip and knee prostheses), were tested for carcinogenic activity following intraarticular administration (20 mg/rat) to groups of 44 male Fischer-344 rats (Charles River Breeding Laboratories, North Wilmington, MA). Control groups received similar intraarticular injections of either a noncarcinogen (manganese powder, negative control rats) or a potent carcinogen (nickel subsulfide powder, positive control rats). The experimental groups of 8-12 rats were observed for 24 months after injection. No local tumors developed at the injection site in the negative control rats or in rats that received the CoCrMo or TiAlV powders; poorly differentiated or pleomorphic sarcomas developed at the injection site in 10 of the 12 positive control rats that were treated with nickel subsulfide. Incidences of primary tumors distant from the injection site did not differ significantly among the experimental groups. This study shows that, under experimental conditions, any carcinogenic activity of CoCrMo or TiAlV wear-debris powders is weak in comparison to nickel subsulfide. Based on this study and observations in other laboratories, intraarticular administration of test materials to rats provides a practical, reliable, and biologically relevant method for carcinogenesis testing of biomaterials used for orthopedic implants.

Improved pulmonary and growth outcomes in cystic fibrosis by newborn screening

Newborn screening for cystic fibrosis (CF) is effective in improving long‐term growth outcomes. However, there is conflicting evidence that early diagnosis maintains normal pulmonary function. Our goal was to determine if newborn screening results in improved longitudinal growth and maintenance of normal pulmonary function.

Effects of nickel chloride on lactating rats and their suckling pups, and the transfer of nickel through rat milk

The excretion of nickel into rat milk following subcutaneous (sc) doses of nickel chloride (NiCl2) and the effects on the lactating rat and her suckling pups were determined. Plasma and milk Ni concentrations increased in a dose-dependent manner 4 hr after single doses of 0, 10, 50, or 100 mumol NiCl2/kg to lactating rats, giving milk/plasma Ni ratios of 0.02. Peak plasma Ni concentrations were reached 4 hr after injection, while milk Ni increased until 12 hr and remained elevated at 24 hr. Dosing for 4 days at 50 or 100 mumol NiCl2/kg/day led to higher milk/plasma Ni ratios of 0.10. These doses of NiCl2 had no effect on body weight but caused decreased food consumption, thymic atrophy, and a small increase in hepatic lipid peroxidation in the dams. Significant alterations in milk composition, which were not due to decreased food consumption as determined in pair-fed rats, included increased milk solids (42%) and lipid (110%), and decreased milk protein (29%) and lactose (61%). NiCl2 treatment also caused significant decreases in mammary RNA content and the RNA/DNA ratio compared to both ad libitum-fed and pair-fed rats, indicating that milk synthetic activity was reduced by NiCl2. Pups suckling the NiCl2-treated dams had plasma Ni concentrations of 24 and 48 micrograms/liter in the 50 and 100 mumol/kg dose groups, respectively, and had decreased liver weight but no changes in hepatic lipid peroxidation or thymus weight. The results indicate that high doses of NiCl2 led to the excretion of Ni into rat milk and changes in milk quality and production. Reductions in liver weight in the suckling pups were also observed which may have been due to nickel exposure or to changes in milk composition.

In vitro characteristics of white cell-reduced single-unit platelet concentrates stored in syringes

BACKGROUND: Platelet concentrates (PCs) for premature infants may be subjected to filtration, centrifugation, and various storage conditions before transfusion.

Cobalt in periprosthetic soft tissue: Observations in 6 revision cases

Cobalt (Co) was analyzed in sera obtained before surgery and in biopsies of periarticular soft tissue from 7 control patients undergoing primary total hip arthroplasty and from 6 Co-exposed patients who developed aseptic loosening of the femoral component after hip arthroplasty (CoCrMo alloy, greater than 59 percent Co, metal-on-plastic type). Serum-Co concentrations were not elevated in the Co-exposed patients compared with control patients or healthy adults. In 5 of the 6 Co-exposed patients, Co concentrations were greatly increased in periprosthetic tissue sections 0-1 mm from the synovial surface (median 2.4 [2.1-27] micrograms Co/g) compared with corresponding sections from the control patients (median 0.4 [0.1-0.6] microgram Co/g). Co concentrations diminished in tissue sections at successive distances of 2-3 and 4-5 mm from the synovial surfaces. In the Co-exposed patients, Co concentrations in sera and periprosthetic soft tissues were not correlated, indicating that serum Co concentration is not a reliable index of the Co burden in periprosthetic soft tissue.

In vitro characteristics of volume‐reduced platelet concentrate stored in syringes

For convenience, small volumes of platelet concentrate (PC) intended for neonatal patients are often dispensed in syringes. The PC, however, may remain in the syringe for up to several hours before the actual transfusion. As there are few data on the effect of such syringe storage on PCs, the in vitro syringe storage properties of small volumes of 1‐ and 5‐day‐old units, and volume‐reduced units of PC were evaluated. In four separate experiments, PCs were stored in syringes in volumes of 10, 15, or 30 mL for up to 6 hours at 20 to 24 degrees C without agitation. Platelets were evaluated for pH, platelet count, and a variety of biochemical and in vitro functional assays. Results showed that even with the equivalent of a full unit of platelets stored in the syringe for up to 6 hours, the pH did not fall below 6.0. Although there was an increase in lactate production and consumption of glucose, which paralleled the decline in pH, the changes were not greater than those seen in platelets stored up to 5 days in gas‐permeable blood bags. Similar results were seen for PCs stored in syringes for 6 hours at 37 degrees C. All of the pH levels recorded at the end of 6 hours of syringe storage were above the minimum required level of pH 6.0. Data from in vitro platelet assays imply that at any time during their shelf life, PCs can be stored in gas‐impermeable polypropylene syringes for up to 6 hours and can maintain acceptable storage characteristics; in vivo data are needed to confirm these observations.

AMPLIFICATION OF GUTHRIE CARD DNA : EFFECT OF GUANIDINE THIOCYANATE ON BINDING OF NATURAL WHOLE BLOOD PCR INHIBITORS

Amplification of DNA from whole blood collected on Guthrie card filter paper presents considerable technical obstacles due to the presence of natural PCR inhibitors (protein, heavy metals, heme, and heme degradation products) and low copy number of genomic material. For this purpose we evaluated guanidine thiocyanate‐impregnated filter paper (GT‐903), a DNA collection device designed specifically to bind PCR inhibitors and preserve DNA in an aqueous extractable form. Compared to standard 903, which retains DNA and elutes inhibitors during aqueous extraction, we found GT‐903 retained 90% of protein, hemoglobin, and iron. SDS‐PAGE analysis indicated that the majority of the protein released from standard 903 corresponded to albumin (70‐) and globin (15‐kDa); negligible levels of these proteins were eluted from GT‐903. To evaluate PCR efficiency, we amplified the 491 bp region encoding the cystic fibrosis ΔF508 mutation. Using comparable template, we found GT‐903 amplification more efficient than standard 903 following qualitative (TBE‐PAGE) and quantitative (anti‐dsDNA EIA) determination. We conclude that GT‐903 provides a good DNA collection device and addresses the complications associated with natural PCR inhibitors. J. Clin. Lab. Anal. 11:87–93.