Genetically predicted circulating levels of copper and zinc are associated with osteoarthritis but not with rheumatoid arthritis.

OBJECTIVE: Osteoarthritis (OA) and rheumatoid arthritis (RA) are both debilitating diseases that cause significant morbidity and disability globally. This study aims to investigate the causal effects of varying blood levels of five minerals -- iron, zinc, copper, calcium, and magnesium, on OA and RA. DESIGN: We performed two-sample Mendelian randomization (MR) analyses to assess the associations of five circulating minerals with OA and RA. Single nucleotide polymorphisms (SNPs) serving as genetic instruments for the circulating mineral levels were selected from large genome-wide association studies of European-descent individuals. The associations of these SNPs with OA and RA were evaluated in UK Biobank participants. Multiple sensitivity analyses were applied to detect and correct for the presence of pleiotropy. RESULTS: Genetically determined copper and zinc status were associated with OA, but not with RA. Per standard deviation (SD) increment in copper increases the risk of OA (OR = 1.07, 95% CI: 1.02-1.13) and one of its subtypes, localized OA (OR = 1.08, 95% CI: 1.03-1.15). Per SD increment in zinc is positively associated with risks of OA (OR = 1.07, 95% CI: 1.01-1.13), generalized OA (OR = 1.18, 95% CI: 1.05-1.31), and unspecified OA (OR = 1.21, 95% CI: 1.11-1.31). Additionally, per SD increment in calcium decreases the risk of localized OA (OR = 0.83, 95% CI: 0.69-0.98). CONCLUSIONS: Genetically high zinc and copper status were positively associated with OA, but not with RA. Given the modifiable nature of circulating mineral status, these findings warrant further investigation for OA prevention strategies.

Zinc uptake into fibroblasts is inhibited by probenecid.

Cellular zinc transport has not been fully characterized. The role of an anion carrier was investigated by treating normal human fibroblasts, and those carrying a mutation which affects zinc transport, acrodermatitis enteropathica (AE), with the anion carrier inhibitor, probenecid. Zinc uptake (2, 10, or 20 micromol 1(-1) 65zinc) was determined during initial rates of uptake (15 min) following treatment with 0, 10 or 20 mmol 1(-1) probenecid. Probenecid stimulated extracellular zinc binding in normal and AE fibroblasts. Probenecid inhibited the internalization of zinc in normal, but not AE, fibroblasts. Normal fibroblasts exhibited an apparent Km which was reduced by 53% and 44% in the 10 and 20 mmol 1(-1) probenecid treated cells. The Vmax was also reduced in the normal fibroblasts by 51% and 50% in the 10 and 20 mmol 1(-1) probenecid treated cells. The results suggest that a probenecid-sensitive anion carrier is involved in the internalization of zinc in human fibroblasts. The lack of an effect of probenecid on the internalization of zinc in the AE fibroblasts suggests that the mutation involves a probenecid-sensitive anion transport system, and that there may be a secondary mechanism for zinc transport in these cells.

Acute myocardial infarction during pregnancy managed with immediate percutaneous transluminal coronary angioplasty.

BACKGROUND: Acute myocardial infarction, a rare peripartum event, is accompanied by significant maternal and fetal mortality. The greatest maternal mortality is realized when myocardial infarction occurs during the third trimester of pregnancy, but there is very little information about the use of acute interventional therapy during pregnancy. CASE: We present a patient who sustained an acute anterior infarction during the third trimester of pregnancy; she underwent immediate cardiac catheterization and coronary angioplasty within 90 minutes of onset of chest pain for high-grade stenosis of the left anterior descending and diagonal coronary vessels. Maternal and fetal outcome was good. CONCLUSION: Immediate percutaneous transluminal coronary angioplasty in the treatment of acute myocardial infarction during pregnancy is a viable treatment option in selected patients.

Age-dependent influence of dietary zinc restriction on short-term memory in male rats.

Zinc is an essential micro-nutrient involved in numerous physiological functions. The high content of zinc in the hippocampus, coupled with the integral involvement of the hippocampus in memory, strongly implicates zinc in memory processing. The hypothesis of the current study was that dietary zinc restriction influenced short-term memory in postweaned rats, and this influence was age-dependent. Male rats (43 days to 18 months old) were divided into five experimental groups based on age, and fed zinc-adequate (zinc at 20 mg/kg as zinc chloride) or zinc-deficient (zinc less than 1-2 mg/kg) diets for a minimum of 3 weeks. Short-term memory was assessed using the distal-cue version of the Morris water maze (MWM). All rats fed the zinc-restricted diet exhibited cyclic anorexia, decreased weight gain, and significantly lower liver and femur zinc concentrations compared to age-matched controls. Further, whole brain, hippocampal, and cerebral wet weights were significantly reduced in the zinc-restricted treatment groups of all the age groups. Only zinc-restricted rats that were less than 62 days of age at the start of zinc restriction demonstrated significantly prolonged escape latencies in the water maze, indicating deficits in short-term memory. Regression analyses confirmed that the short-term memory deficits were correlated with significantly lower hippocampal and cerebral zinc concentrations compared to age-matched control and pair-fed rats. These results emphasize the significance of a critical age of influence for dietary zinc in memory processing, and the importance of considering age when studying zinc nutriture and CNS function.

Water maze performance and changes in serum corticosterone levels in zinc-deprived and pair-fed rats.

The aims of the present study were (1) to evaluate the learning and short- and long-term memory of zinc-deprived (ZD) and pair-fed (PF) rats in a Morris water maze (MWM) and (2) to monitor the serum corticosterone levels of these rats before and after swimming. Young Sprague-Dawley rats (aged 27-31 days) consumed AIN-93G diet for 10 days, and then were separated into ad libitum control (CT), PF and ZD groups. The zinc content of the diet was 25-30 ppm (CT and PF) or <1 ppm (ZD). After 17 days on experimental diets, a MWM was used to test spatial cognition. Delayed-matching-to-place (DMP) test results indicate that both zinc deprivation and food restriction had no effect on short-term memory. The PF rats exhibited significantly impaired learning and thigmotaxia (i.e., wall hugging) in the learning test. The PF group also demonstrated less preference for the target zone in the first 15 s of the probing test. When the total 120 s of the probing test was considered, there were no differences in preference for the target zone, but thigmotaxia was greater in the PF than the CT group. The only behavioral change of the ZD group was thigmotaxia observed during the 120-s probing test following training, indicating the increment of anxiety. Morning basal corticosterone levels before swim training were significantly elevated in the PF group on Day 15 of dietary treatment, whereas a significant elevation of the basal corticosterone level in the ZD group was not statistically significant until Day 22. The data indicate an association between impaired learning, poor searching strategy and elevated corticosterone in the PF group. In contrast, the ZD rats showed normal cognitive performance but had elevated corticosterone and increased anxiety-like behavior (thigmotaxia).

The effect of the acrodermatitis enteropathica mutation on zinc uptake in human fibroblasts.

The acrodermatitis enteropathica (AE) mutation affects intestinal zinc absorption. Our goal was to determine whether the AE mutation affects zinc uptake in human fibroblasts. Zinc uptake was determined during initial rates of uptake (10 min) following incubation in HEPES/saline buffer. Zinc uptake (from 0.25 to 1 microM) into normal fibroblasts was significantly greater than into the AE fibroblasts (p < 0.05). In order to identify factors that may alter cellular zinc uptake and be affected by the AE mutation, zinc uptake in the presence of albumin or bicarbonate was measured. Albumin restricted zinc uptake in both normal and AE fibroblasts, whereas bicarbonate stimulated zinc uptake in the normal fibroblasts. The effect of bicarbonate on zinc uptake in the AE fibroblasts was significantly reduced in both the Pronase-sensitive and Pronase-resistant compartments. Following loading of the fibroblasts with 1 microM zinc for 60 min, zinc efflux and retention were measured. The AE mutation did not affect zinc retention compared to normal fibroblasts. We conclude that the AE mutation affects both zinc binding to the cell surface and its translocation across the plasma membrane into the cell, possibly mediated through a defective anionic exchange mechanism.

Nystatin affects zinc uptake in human fibroblasts.

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 micrograms nystatin/mL or 0.1% dimethylsulfoxide for 10 min at 37 degrees C. The cells were then incubated with 1 to 30 microM 65zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced the K(m) 56% and the Vmax 69%. In the AE fibroblasts, nystatin treatment significantly reduced the Vmax 59%, but did not significantly affect the K(m). The AE mutation alone affected the Vmax for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction in Vmax compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also affects zinc transport by reducing zinc transport.

Partial characterization of a human zinc-deficiency syndrome by differential display.

The effect of the acrodermatitis enteropathica mutation (AE) on gene expression was investigated using differential display. Two differentially expressed cDNAs were partially characterized. The NA8 cDNA (HT11A anchor and HAP 8 random primer pair) was expressed in greater quantity in normal fibroblasts, was 249 bp, and hybridized to three mRNA species (2 kb, 1 kb, 0.8 kb). Northern blot analysis indicated that the relative amounts of the AE mRNA species were reduced by 73%, 75%, and 52%, respectively. The cDNA sequence exhibited 92-93% homology to the human cytochrome oxidase subunit II, as analyzed through the GenBank database. The AEG4 cDNA species (HT11G anchor and HAP 4 random, primer pair) was expressed in greater quantity in AE fibroblasts, was 197 bp, and hybridized to two mRNA species (9 kb, 4 kb). Northern blot analysis indicated that the 9-kb mRNA species was present equally in AE and normal cells, but the 4-kb mRNA species was only present in the AE fibroblasts. The cDNA sequence exhibited 92% homology to LINE1 human retrotransposons, as analyzed through the GenBank database. The functional relationship between the mutation and the reduced expression of cytochrome oxidase subunit II is unknown at this time and needs to be addressed. The increased expression of the LINE1 element in AE fibroblasts may be indicative of an insertion mutation affecting the mRNA of a protein involved in zinc transport, a prospect which requires further investigation.

Differences in the cellular zinc content and 5'-nucleotidase activity of normal and acrodermatitis enteropathica (AE) fibroblasts.

The acrodermatitis enteropathica (AE) mutation affects zinc (Zn) metabolism in human fibroblasts. We hypothesize that the mutation affects the cell Zn content, which subsequently affects the activity of various zinc-dependent enzymes, such as 5'-nucleotidase. Therefore, normal and AE fibroblasts were grown in normal medium containing physiological levels of Zn (16 micromol/L) for approximately 24 h. The medium was replaced by normal medium (16 micromol/L Zn), Zn-depleted medium (1.5 micromol/L Zn), or Zn-supplemented medium (200 micromol/L Zn) for another 24 h. Regardless of the Zn concentration of the growth medium, the AE fibroblasts contained significantly less Zn than normal fibroblasts grown in comparable medium. Nevertheless, growth of the fibroblasts in 200 micromol/L Zn medium significantly increased the cell Zn content fourfold of both normal and AE fibroblasts. The activity of 5'-nucleotidase in the AE fibroblasts grown in 16 micromol/L Zn or 1.5 micromol/L Zn medium was also significantly lower than in normal fibroblasts. Changing the growth medium from 16 micromol/L Zn to 1.5 micromol/L Zn medium did not affect the activity of the enzyme in either genotype. Cells grown in 200 micromol/L Zn medium exhibited threefold greater 5'-nucleotidase activity in AE fibroblasts, but had no affect on enzyme activity in normal cells. In summary, altering the cell Zn content of normal fibroblasts did not result in a significant change in their 5'-nucleotidase activity. However, AE fibroblasts grown in 200 micromol/L Zn medium exhibited recovery of their 5'-nucleotidase activity to normal levels. These results support the hypothesis that the AE mutation affects the cellular Zn content. The lower cell Zn content subsequently affects the activity of 5'-nucleotidase.

Priming with magnesium-deficient media inhibits preadipocyte differentiation via potential upregulation of tumor necrosis factor-alpha.

The effect of priming stromal-vascular cells in primary cultures with magnesium-deficient (MgD) media on preadipocyte differentiation was studied. Cultures were derived from dorsal subcutaneous fat tissue of young pigs and maintained 3 d in serum-free control or MgD Dulbecco's modified Eagle's medium, 3 d in 10% fetal bovine serum and dexamethasone, and 6 d in insulin. At d 12 of culture, immunocytochemical and glycerophosphate dehydrogenase assays indicated depressed adipocyte differentiation in the MgD groups. Cultures were enriched for preadipocytes up to 50% of total cells. On the third day of treatment with control and MgD medium, total cell lysates were isolated and 50 microg of them were run on two-dimensional gel electrophoresis. The separated proteins from both treatment groups showed similar patterns. However, spots of proteins with predicted molecular weight in the range of 25.8-37.4 kDa and pI of 5.39-5.85 were sixfold denser from the MgD 10 groups than from the controls. These proteins migrate similarly to tumor necrosis factor-alpha (TNF-alpha). The amount of TNF-alpha in cell lysates from the MgD group was about 2.35 times greater than controls determined by TNF-alpha-ELISA. It is likely that proteins upregulated by MgD medium are TNF-alpha isoforms.

The acrodermatitis enteropathica mutation transiently affects zinc metabolism in human fibroblasts.

The acrodermatitis enteropathica (AE) mutation has been shown to affect zinc transport in human intestinal biopsies. However, whether the mutation is also expressed in human fibroblasts has not been determined. The activity of the zinc-dependent enzyme, 5' nucleotidase, and cell zinc content were measured in normal and AE fibroblasts 2 and 4 d after subculturing to determine the effect of the AE mutation on zinc metabolism. The activity of 5' nucleotidase in AE cells was 68% of normal at 2 d after subculturing. Although 5' nucleotidase activity had decreased significantly in both normal and AE fibroblasts at 4 d after subculturing, there was no significant difference between the two genotypes. The zinc content of AE fibroblasts was also significantly reduced. Acrodermatitis enteropathica fibroblasts contained 62% less zinc than normal fibroblasts at 2 d. By 4 d the normal fibroblast zinc content had decreased to that of the AE fibroblasts. The uptake and transport of 65Zn into AE fibroblasts at 2 d was measured because these cells exhibited reduced 5' nucleotidase activity and cell zinc content at this time. The uptake of zinc over a 90-min time period was the same in the two genotypes. However, AE fibroblasts incubated with 2-10 mumol Zn/L for 15 min had significantly slower zinc transport compared with normal fibroblasts. In both genotypes, Michaelis-Menten kinetics were observed. Normal and AE fibroblasts had similar affinities for zinc (Km), but AE fibroblasts exhibited a Vmax which was reduced by 38%. These results indicate that the phenotypic expression of the AE mutation occurs in a time-dependent manner, is not restricted to the intestine and is also transiently expressed in human fibroblasts, resulting in abnormal zinc metabolism in these cells.

Intestinal metallothionein in lethal-milk mice with systemic zinc deficiency.

Lethal-milk (C57BL/6J-lm) mice over 12 months of age exhibit clinical signs of systemic Zn deficiency. Such lm mice have increased concentrations of metallothionein (MT) in the intestinal mucosa. Various concentrations of Cd or Zn were added to the drinking water. MT was assayed using the Cd-saturation/hemolysate method and for sulfhydryl concentration (MT has 33% cysteine residues) with Ellman's reagent. As assayed by both methods, mucosa from untreated lm mice contained approximately twice as much MT as did the C57BL/6J-(+lm/+lm) (B6) controls. Treatment with 150 and 500 ppm Zn removed the genotypic differences observed for the untreated and Cd-treated mice. These results are consistent with the lm mutation affecting Zn metabolism through impaired MT metabolism as measured for the intestinal mucosa. These studies do not eliminate the possibility that the liver may also contribute.

Zinc metabolism in lethal-milk mice. Otolith, lactation, and aging effects.

Lethal-milk (lm), a recessive mutation, occurred in the C57BL/6J inbred strain of mice. Lactating lm dams produce a zinc-deficient milk that is lethal to all nursing pups. If foster-nursed on normal dams, lm pups survive and become reproductively mature. Injection of zinc-glycinate into the pups or zinc supplementation of the water of the lactating dams reduces lethality. Other pleiotropic effects in lm mice include congenital otolith defects with delayed righting, "tail-spinning," and abnormal swimming. These effects are diagnostic criteria for segregation of lm mice among backcross progeny. About 40 percent of the expected number of lm pups survive to weaning. Zinc supplementation of the dam improves development of saccular but not of utricular otoliths; zinc does not improve survival of the lm pups among backcross progenies. The lm mice over eight months of age also exhibit extensive hair loss, dermatitis, and skin lesions. Possible roles of metallothionein in zinc and copper metabolism are discussed in regard to the pleiotropic effects of the lethal-milk mutation.

The acrodermatitis enteropathica mutation affects protein expression in human fibroblasts: analysis by two-dimensional gel electrophoresis.

The acrodermatitis enteropathica (AE) mutation affects zinc uptake in human fibroblasts. However, the specific biochemical lesion has not been identified. We have used the technique of two-dimensional gel electrophoresis to identify protein differences in total cell lysate isolated from normal and AE fibroblasts. Two proteins with estimated molecular weights of 49.6 and 49.9 kDa and an isoelectric point of 5.1 were identified in normal fibroblasts but absent from AE fibroblasts. The proteins were purified, subjected to in-gel trypsin digest and the resulting peptides separated by HPLC. Sequences from three peptide fragments (8, 15 and 18 amino acids) were obtained after Edman degradation. None of the fragments exhibited homology to any amino acid sequences in the nonredundant Genbank database. The 15 and 18 amino acid fragments each exhibited 100% homology to a 136 amino acid expressed sequence tag that was homologous (43%) to adipophilin. However, the 15 and 18 amino acid fragments were only 30 and 44% homologous, respectively, to corresponding regions within the expressed sequence tag. Therefore, the 49.6/49.9 kDa protein absent from AE fibroblasts was not related to adipophilin. The 8 amino acid fragment did not exhibit homology to any expressed sequence tag. Therefore, the 49.6/49.9 kDa proteins are novel and may be the cause of the reduced zinc uptake and abnormal zinc metabolism characteristic of fibroblasts carrying the AE mutation.

Erythrocyte metallothionein as an index of zinc status in humans.

Metallothionein concentrations in erythrocyte lysates derived from human subjects were measured by an ELISA procedure. IgG obtained from serum of sheep injected with human metallothionein 1 was used in this competitive assay. Subjects were fed a semipurified zinc-deficient diet (0.7 mg of zinc per kg of diet) for an 8-day depletion period after 3 days of acclimation. Fasting plasma zinc concentrations were reduced approximately 7%. Metallothionein in the erythrocyte lysates was significantly decreased to 59% of the initial level by the end of the depletion period. Supplementation of these depleted subjects with zinc (50 mg) did not increase erythrocyte metallothionein levels within 24 hr. Daily supplementation of control subjects with zinc (50 mg/day) increased erythrocyte metallothionein to a 7-fold maximum within 7 days. These levels were reduced by 61% within 14 days after zinc supplementation was terminated. Incubation of rat [35S]metallothionein with human erythrocyte lysate showed a time-dependent increase in 35S soluble in 20% trichloroacetic acid, indicating degradation of the labeled protein, presumably via protease activity in the lysate. It is proposed that zinc supplementation induces erythrocyte metallothionein during erythropoiesis and that low zinc intake decreases synthesis and/or accelerates degradation of the protein in reticulocytes/erythrocytes. Metallothionein levels in erythrocytes may provide a useful index upon which to assess zinc status in humans.

Treatment of Wilson's disease with zinc: X. Intestinal metallothionein induction.

Oral zinc therapy is effective in controlling copper balance in patients with Wilson's disease and blocks the intestinal absorption of copper, as demonstrated by uptake of copper 64 and copper balance measurements. In this study, 64Cu uptake measurements were concomitantly carried out with intestinal biopsies to investigate the relationship of reduced copper absorption to the levels of intestinal metallothionein in patients with Wilson's disease at different stages of zinc therapy. A pronounced increase in intestinal metallothionein levels and a sharp drop in 64Cu absorption were found 4 to 5 days after the initiation of zinc treatment. Conversely, metallothionein levels decreased and 64Cu uptake increased on the discontinuation of zinc therapy. The data indicate that 64Cu absorption varies as a function of intestinal metallothionein level. Intestinal metallothionein levels were found to correlate linearly with urinary zinc levels, which reflect body zinc status. These findings support our hypothesis that intestinal metallothionein induction mediates decreased copper absorption observed during zinc therapy. The suppressive effect of zinc on copper absorption appears to have a half-life of about 11 days.

Supplementation with L-histidine during dietary zinc repletion improves short-term memory in zinc-restricted young adult male rats.

Zinc, an essential dietary element, modulates neurotransmission in brain regions associated with cognition. Cognitive dysfunction has been reported in offspring of female rats fed zinc-restricted diets during gestation and/or lactation. Studies on the cognitive effects of zinc restriction during young adulthood are limited. After a 3-wk period of dietary zinc restriction, male rats (71-75 d old) were repleted with zinc chloride alone, or zinc chloride supplemented with L-histidine, and short-term memory was measured using the Morris water maze. During restriction, zinc-restricted rats demonstrated significantly longer (86.0%) retrieval latencies than nonrestricted controls, and significantly lower liver (25.5%), bone (32.5%) and hippocampal (3.2%) zinc concentrations. During subsequent repletion, rats repleted with zinc chloride supplemented with L-histidine improved their retrieval latencies to the extent that they were no longer significantly different from controls by repletion d 3. This was associated with a return of hippocampal zinc concentrations to control values by repletion d 3. The mean retrieval escape latencies of the zinc chloride-repleted rats remained significantly prolonged (75.0%). Collectively, these data indicate the following: 1) feeding a zinc-restricted diet for 3 wk impairs short-term memory in young adult male rats, and 2) repletion with dietary zinc supplemented with L-histidine improves short-term memory function more efficiently than dietary zinc chloride alone. The latter point suggests that dietary zinc supplemented with L-histidine is more bioavailable to the brain than zinc provided as zinc chloride alone. These findings are important in that they highlight the importance of both dietary zinc formulation and the use of functional assessments in determining zinc nutriture.

Enzyme-linked immunosorbent assay for human metallothionein: correlation of induction with infection.

Very little information is available concerning the relationship between metallothionein and disease in humans. Recently, investigators have used the Cd-heme method to measure metallothionein levels in human liver samples obtained from autopsy. This assay, however, is not sensitive enough to measure metallothionein in small tissue biopsy specimens. As an alternative, we report the development of a human metallothionein enzyme-linked immunosorbent assay (ELISA). This assay used high-performance liquid chromatography-purified human metallothionein-1 and purified sheep anti-human metallothionein-1 IgG. A limiting antigen coating concentration of 100 ng/ml and a minimal antibody dilution of 1:4000 were chosen. The sensitivity of the ELISA extended to 300 ng/ml (15 ng). The coefficients of interassay and intraassay variation were 15.4% and 4.2%, respectively. Human livers obtained from autopsy were assayed by this method and the values compared with values obtained by the Cd-heme method. The livers were separated by their autopsy reports into four groups: normal, immunosuppression, cancer, and infection. Livers from the infection group (ELISA 2979 micrograms/gm, Cd-heme 1201 micrograms/gm) contained significantly more metallothionein than those from the normal (ELISA, 1035 micrograms/gm, Cd-heme 245 micrograms/gm) and the immunosuppression (ELISA 1272 micrograms/gm, Cd-heme 221 micrograms/gm) groups (p less than 0.05). The cancer group (ELISA 2415 micrograms/gm) also had significantly elevated liver metallothionein levels. We conclude that this ELISA is sensitive enough for the measurement of tissue samples. Furthermore this assay is comparable to the Cd-heme assay in its ability to reflect metallothionein values among various treatment groups. We postulate that hepatic metallothionein induction is mediated by disease-related mechanisms such as interleukin-1, glucocorticoid secretion, or both.