Effect Of ZA Research Articles

The Effect of Zoledronic Acid on Serum Biomarkers among Patients with Chronic Low Back Pain and Modic Changes in Lumbar Magnetic Resonance Imaging.

The aim of the current study was to compare changes in serum biomarkers, including inflammatory mediators, signaling molecules, growth factors and markers of bone turnover after a single intravenous infusion of 5 mg zoledronic acid (ZA, a long-acting bisphosphonate; n = 20) or placebo (n = 20) among patients with Modic changes (MC) and chronic low back pain in a randomized controlled design. The MCs were classified into M1, predominating M1, predominating M2, and M2. We measured the serum concentrations of 39 biomarkers at baseline, and one month and one year after treatment. After Benjamini–Hochberg (B–H) correction, we observed significant differences in three biomarkers over one year: Interferon-γ-inducible protein (IP-10) had risen in the ZA group (p = 0.005), whereas alkaline phosphatase (AFOS) and intact procollagen I N-terminal propeptide (iPINP) had significantly decreased in the ZA group, but had not changed in the placebo group (p < 0.001 for both). Change in iPINP correlated with change in the volume of all MC and M1 lesions. ZA downregulated bone turnover markers as expected and, surprisingly, increased the chemokine IP-10 relative to placebo treatment. This adds to our knowledge of the effects of ZA on MC and the biomarkers that signal this process.

Zoledronic acid blocks the interaction between breast cancer cells and regulatory T-cells

BackgroundZoledronic acid (ZA), a nitrogen-containing bisphosphonate, inhibits osteoclastogenesis. Emerging evidence suggests that ZA has anti-tumor and anti-metastatic properties for breast cancer cells. In a mouse model of ZA-related osteonecrosis of the jaw, ZA administration was found to suppress regulatory T-cells (Tregs) function. Our previous reports also demonstrated ZA acted as an immune modulator to block Tregs. Manipulation of Tregs represents a new strategy for cancer treatment. However, the relationship among ZA, Tregs, and cancer cells remains unclear. In this study, we investigated the effects of ZA on the interaction of breast cancer cells and Tregs.MethodsThe anti-tumor effect of ZA on triple negative breast cancer cell lines were validated by XTT, wound healing and apoptosis analysis. A flow cytometry-based assay was used to analyze the immunosuppressive effect of Tregs treated with media conditioned by breast cancer cells, and a transwell assay was used to evaluate the chemotactic migration of Tregs. Differential gene expression profile on MDA-MB-231 treated with ZA (25 μM) was analyzed by. microarrays to describe the molecular basis of actions of ZA for possible direct anti-tumor effects. Enzyme-linked immunosorbent assays and quantitative real-time PCR were used to investigate the effect of ZA on the expression of cytokines/factors by breast cancer cells.ResultsZA was found to inhibit the proliferation and migration of breast cancer cells. Media conditioned by the MDA-MB-231 cells promoted the expansion, chemotactic migration, and immunosuppressive activity of Tregs, and these effects were attenuated in a dose-dependent manner by ZA treatment, and the attenuation was due to reduced expression of selected breast cancer cell factors (CCL2, CCL5, and IDO).ConclusionsZA can significantly affect the interaction between breast cancer cells and Tregs. Our findings indicate that ZA is a potential therapeutic agent that can be used to reduce cancer aggressiveness by abolishing the supportive role of Tregs.

The effects of group size and subtherapeutic antibiotic alternatives on growth performance and morbidity of nursery pigs: a model for feed additive evaluation.

The objectives of this experiment were to evaluate the effects of alternatives to antibiotic growth promoters (AGP), two group sizes, and their interaction on nursery pig performance to serve as a model for future AGP alternative studies. A 41-d experiment was conducted in a commercial wean-to-finish barn; 1,300 piglets weaned at 21 d of age (weaned 2 or 4 d prior to experiment; 6.14 ± 0.18 kg BW; PIC 1050 sows and multiple sire lines) were blocked by sire, sex, and weaning date, then assigned to eight treatments: four dietary treatments each evaluated across two group sizes. The four dietary treatments were: negative control (NC), positive control (PC; NC + in-feed antibiotics), zinc oxide plus a dietary acidifier (blend of fumaric, citric, lactic, and phosphoric acid) (ZA; NC + ZnO + acid), and a Bacillus-based direct-fed-microbial (DFM) plus resistant potato starch (RS) (DR; NC + DFM + RS). The two group sizes were 31 or 11 pigs/pen; floor space was modified so area/pig was equal between the group sizes (0.42 m2/pig). There were 7 pens/diet with 11 pigs/pen and 8 pens/diet with 31 pigs/pen. Data were analyzed as a randomized complete block design with pen as the experimental unit. Diagnostic assessment of oral fluids, serum, and tissue samples was used to characterize health status. Pigs experienced natural challenges of acute diarrhea and septicemia in week 1 and porcine reproductive and respiratory syndrome virus (PRRSV) in weeks 4–6. There was a significant interaction between diet and group size for ADG (P = 0.012). PC increased ADG in large and small groups (P < 0.05) and ZA increased ADG only in large groups (P < 0.05). Small groups had improved ADG compared to large groups when fed NC or DR diets (P < 0.05). Similarly, PC increased ADFI (P < 0.05). Compared to NC, ZA improved ADFI in large groups only (P < 0.05; diet × group size: P = 0.015). Pigs fed PC had greater G:F than NC (P < 0.05), and small groups had greater G:F than large groups (P < 0.05). There was no effect of ZA or DR on G:F. Pigs fed PC required fewer individual medical treatments than NC and pigs fed ZA were intermediate (P = 0.024). More pigs were removed from large than small groups (P = 0.049), and there was no effect of diet on removals (P > 0.10). In conclusion, careful study design, protocol implementation, sample collection, and recording of important information allowed us to characterize the health status of this group of pigs and determine treatment effects on growth performance and morbidity.

The effect of vitamin D and zoledronic acid in bone marrow adiposity in kidney transplant patients: A post hoc analysis.

PurposeOsteoblasts and adipocytes are derived from mesenchymal stem cells. An imbalance in the differentiation of these lineages could affect the preservation of bone integrity. Several studies have suggested the importance of this imbalance in the pathogenesis of osteoporosis after kidney transplant (KT), but the role of bone marrow adiposity in this process is not well known, and if the treatment with the anti-absorptive (zoledronic acid-ZA) drugs could attenuate bone loss. Thus, our objective was compare bone marrow adiposity, osteoblasts and osteocytes before and after KT, verify an association between bone remodeling process (Turnover, Volume, and Mineralization—TMV classification), the osteocyte sclerostin expression to evaluate if there is a role of Wnt pathway, as well as the effect of ZA on these cells.MethodsWe studied 29 new living-donor KT patients. One group received ZA at the time of KT plus cholecalciferol for twelve months, and the other group received only cholecalciferol. Bone biopsies were performed at baseline and after 12 months of treatment. Histomorphometric evaluation was performed in bone and bone marrow adipocytes. Sclerostin (Scl) expression in osteocytes was evaluated by immunohistochemistry.ResultsSome bone marrow adiposity parameters were increased before KT. After KT, some of them remained increased and they worsened with the use of ZA. In the baseline, lower bone Volume and Turnover, were associated with increased bone marrow adiposity parameters (some of them). After KT, both groups showed the same associations. Osteocyte Scl expression after KT decreased with the use of ZA. We observed also an inverse association between bone adiposity parameters and lower osteocyte sclerostin expression 12 months after KT.ConclusionIn conclusion, the present study suggests that KT fails to normalize bone marrow adiposity, and it even gets worse with the use of ZA. Moreover, bone marrow adiposity is inversely associated with bone Volume and Turnover, which seems to be accentuated by the antiresorptive therapy.

Zoledronic acid induces micronuclei formation, mitochondrial-mediated apoptosis and cytostasis in kidney cells

AimsZoledronic acid (ZA), a FDA approved drug has used widely in the treatment of bone metastasis complications, has been linked to renal toxicity with unclear mechanism. The present study is aimed at investigating the genotoxic and cytotoxic effects of ZA in renal epithelial cells. Main methodsThe genotoxic effect of ZA in Vero and MDCK cells determined by cytokinesis block micronucleus (CBMN) assay. The cytotoxic effect assessed by analysing cell cycle profile, cell death and mitochondrial membrane potential by flow cytometry using propidium iodide, AnnexinV-FITC/PI and JC1 dye staining, respectively, BAX and Bcl-2 expression by Western blotting and caspase activity by spectrofluorimetry. Key findingsThe cytotoxic effect of ZA based on MTT assay revealed variable sensitivities of Vero and MDCK cells, with IC50 values of 7.41 and 109.58 μM, respectively. The CBMN assay has shown prominent dose-dependent (IC10-50) induction of micronuclei formation in both cells, indicating ZA's clastogenic and aneugenic potential. Further, the ZA treatment led the cells to apoptosis, evident from dose-dependent increase in the percentage of cells in subG1 phase and display of membranous phosphatidylserine translocation. Studies also confirmed apoptosis through mitochondria, evident from the prominent increase in BAX/Bcl-2 ratio, mitochondrial membrane depolarization and caspase-3/7 activity. In addition, ZA reduces cytokinetic activity of renal cells, evident from dose-wise lowered replicative indices. SignificanceThe study depict ZA's potential genotoxic effect along with cytotoxic effect in renal epithelial cells, could be key factors for the development of renal complications associated with it, which prompts renal safety measures in lieu with ZA usage.

The inhibitory effect of zoledronate on early-stage osteoinduction by recombinant human bone morphogenetic protein 2 in an osteoporosis model

This study evaluated the effect of the combined treatment of intravenous zoledronic acid (ZA, 0.08 mg/kg) and rhBMP-2 (5 µg) on osteogenesis in a calvarial defect model of ovariectomized SD rats. New bone formation was evaluated 4 or 8 weeks after calvarial defect implantation using micro-CT and histology. Micro-CT results revealed that the rhBMP-2 group showed significantly higher calvarial defect coverage ratio compared with the ZA + rhBMP-2 group at 4 weeks. In addition, bone formation indices were significantly lower in ZA + rhBMP-2 group when compared with the rhBMP-2 group after 4 weeks, which indicates a negative effect of ZA on the initial bone formation and the bone quality. At 8 weeks, the negative effect induced by ZA treatment was alleviated as time passed. Histological examination showed similar results to the micro-CT measurements. In conclusion, although ZA treatment lowered the new bone formation induced by rhBMP-2 initially, as time passed, the negative effect was decreased.

Zoledronic Acid Improves Overall Survival Compared To Pamidronate In Multiple Myeloma

BackgroundRecent practice guideline changes suggest all patients with MM should receive bisphosphonates, regardless of the presence of bone disease. Nitrogen-containing bisphosphonates, ZA and PAM, inhibit protein prenylation, a process crucial for osteoclast survival. This inhibition also interferes with tumor cell adhesion, angiogenesis, and proliferation suggesting potential for anti-tumor activity of bisphosphonates. Morgan et al showed increased OS with use of ZA versus clodronate in MM. This increase was independent of the effect on bone disease, supporting anti-tumor activity of ZA. ZA and PAM are the two approved bisphosphonates for MM in the United States. In in-vitro studies, ZA has increased potency compared to PAM, a finding that may result in increased apoptosis of tumor cells. A meta-analysis by Mhaskar et al demonstrated an association between higher bisphosphonate potency and improved OS in MM. In an effort to clarify the effect of ZA versus PAM on OS in MM, we evaluated outcomes in a large cohort of United States Veterans with MM. MethodsWe identified 1,018 patients with newly diagnosed MM in the Veterans Administration Cancer Registry between 2002 and 2009, who were treated with either PAM or ZA, but not both. Data was collected on age, co-morbidities, date of diagnosis and death, myeloma specific and supportive medications, and baseline lab data. Cox proportional-hazards was used to assess association between bisphosphonate use and OS while controlling for other prognostic factors. Propensity score analyses were performed to reduce confounding by indication, using the inverse probability weighting (IPW) approach of Cole and Hernan and propensity score matching. The covariates for each propensity model were the same as those in the main analysis. ResultsOf the 1,018 patients in the cohort, 383 received ZA and 635 received PAM. The median follow-up was 26.9 months. After adjustment using propensity score groups, baseline characteristics were well balanced between the groups (Table 1). Kaplan-Meier curves showing OS of patients receiving ZA versus PAM are shown in Figure 1. After controlling for age, weight, comorbidity score, era of diagnosis (before or after 2006), baseline lab characteristics, and treatment, OS was significantly improved with ZA compare to PAM (HR 0.84; 95% CI, 0.72-0.98). In both the IPW and propensity score matching analyses, ZA significantly improved OS in MM (HR 0.84; 95% CI, 0.72-0.97) and (HR 0.83; 95% CI, 0.69-0.99) respectively.Table 1Baseline CharacteristicsUnadjusted DataAdjusted with IPWPropensity Score MatchingPAMZAP-valuePAMZAP-valuePAMZAP-valueAge (mean)Male Sex (%)9896.10.077397.397.10.830496.696.61.0000Race (%)White and other74.57072.87370.571.6Black25.5300.116927.2270.938129.528.40.7397Mean Charlson score2.62.40.18512.62.50.37212.62.40.3503Transplant (%)15.6110.038513.713.80.959111.411.41.0000Melphalan_use (%)35.336.80.619936.235.50.811238.436.70.6404Lenalidamide_use(%)21.727.40.039423.423.90.853224.727.60.3910Thalidomide_use(%)51.352.70.664351.551.30.939651.152.80.6508Steroid Alone (%)22.716.20.012520.520.80.913319.017.10.4926Bortezomib_use(%)34.332.90.639733.732.90.783634.133.20.8109Hemoglobin (mean)10.710.70.744210.810.60.149310.910.70.2788Creatinine (mean)1.91.80.00121.81.90.53611.51.60.7944Albumin (mean)3.33.30.88033.33.20.65173.33.20.6348Calcium (mean)9.79.50.05819.79.60.56309.79.50.0962 [Display omitted] ConclusionIn this large, multicenter cohort study, ZA improved OS compared to PAM in patients with MM. The benefit persisted even after controlling for known patient and treatment related prognostic factors, as well as controlling for differences in baseline patient characteristics. Bisphosphonates play a key role in the treatment of MM for the prevention of MM bone disease. Evidence suggests that bisphosphonates may also have a direct anti-tumor effect in MM. Our study adds to this growing body of evidence and provides rationale for selecting ZA over PAM in most patients with MM. Disclosures:No relevant conflicts of interest to declare.

Abstract 3715: Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma.

Abstract Zoledronic acid (ZA), a third generation of bisphosphonates, is known to have both anti-osteolytic and anti-tumor activities. It exerts anti-osteolytic function by interfering with mevalonate pathway and preventing protein prenylation required for growth and survival in osteoclast cells. Also, similar function has been found to induce cancer apoptosis and inhibit cancer growth in bone-related cancer, but its effect on other cancer types, including head and neck squamous cell carcinoma (HNSCC) is still unclear. In this study, we first monitored the effect of ZA on cancer growth in HNSCC cell lines in vitro. ZA significantly suppressed cancer growth, shown by decreased anchorage independent growth in soft agar assay and diminished cell growth in organotypic raft culture. Because cancer stem cell (CSC; also called tumor-initiating cell) plays a significant role in cancer growth, we further investigated the effect of ZA on CSC in HNSCC. ZA-treated HNSCC cells significantly decreased CSC phenotypes such as sphere forming ability and migratory ability. In SCC4, a tongue squamous cell carcinoma cell line, side population and expression of pluripotency marker genes were significantly decreased by ZA. Also, ZA inhibited the CSC-related Sonic Hedgehog pathway in SCC4 as demonstrated by decreased mRNA expression of transcriptional factor, Gli-1. Taken together, our data suggest that ZA targets CSC and hence inhibits cancer growth in HNSCC. Citation Format: Sung Hee Lee, Reuben Kim, Mo Kang, No-Hee Park, Ki-Hyuk Shin. Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3715. doi:10.1158/1538-7445.AM2013-3715

The effects of combined human parathyroid hormone (1-34) and zoledronic acid treatment on fracture healing in osteoporotic rats

Ovariectomized (OVX) rats with tibial fracture received vehicle, ZA, PTH, or ZA plus PTH treatment for 4 and 8 weeks. Bone metabolism, callus formation, and the mass of undisturbed bone tissue were evaluated by serum analysis, histology, immunohistochemistry, radiography, micro-computerized tomography, and biomechanical test. Previous studies have demonstrated the effect of ZA or PTH on osteoporotic fracture healing. However, reports about effects of ZA plus PTH on callus formation of osteoporotic fracture were limited. This study was designed to investigate the impact of combined treatment with ZA and PTH on fracture healing in OVX rats. Twelve weeks after bilateral ovariectomy, all rats underwent unilateral transverse osteotomy on tibiae. Animals then randomly received vehicle, ZA (1.5 μg/kg weekly), PTH (60 μg/kg, three times a week), or ZA plus PTH until death at 4 and 8 weeks. The blood and bilateral tibiae of rats were harvested for evaluation. All treatments increased callus formation and strength other than the control; ZA + PTH showed the strongest effects on percent bone volume (BV/TV), trabecular thickness, total fluorescence-marked callus area, and biomechanical strength. Additionally, inhibited RANKL and enhanced osteoprotegerin expression were observed in the ZA + PTH group. But no difference in bone mineral density and BV/TV of the contralateral tibiae was observed between treated groups. Findings in this study suggested an additive effect of ZA and PTH on fracture healing in OVX rats, and this additive effect was specific to callus formation, not to undisturbed bone tissue.

Abstract 565: Effects of zoledronic acid on the interaction between mesenchymal stem cells and breast cancer cells

Abstract Zoledronic acid (ZA) is the mainstay of treatment of bone metastases associated with a wide variety of tumors, including breast cancer. It has been recently shown that ZA increases the progression free-survival of estrogen receptor (ER)-positive breast cancer patients by reducing both loco-regional and distant metastases, suggesting that this compound might act directly on breast cancer cells. However, ZA very rapidly concentrates in the bone following intravenous administration, and therefore this drug is unlikely to have a direct effect on cell populations outside the bone. Recent reports have shown that bone-marrow-derived mesenchymal stem cell (MSCs) are recruited to the stroma of developing tumors where they increase the metastatic potential of breast cancer cells by secreting cytokines, chemokines and growth factors that sustain breast cancer migration, invasion and metastasis. In particular, the chemokine CCL5 (RANTES) plays an important role in this phenomenon. Therefore, we investigated the effects of ZA on the ability of primary MSCs to secrete cytokines and growth factors that might be involved in breast cancer progression. We found that treatment with ZA produced marginal effects on the growth of human primary MSCs with an approximately 25% growth inhibition at 20 μM. In contrast, treatment with similar concentrations of ZA almost completely suppressed the ability of MSCs to secrete RANTES (&amp;gt;90% reduction). The effect of ZA on RANTES was quite specific, since marginal inhibition of the secretion of different angiogenic growth factors, such as VEGF, IL-8 and bFGF was observed. Treatment of MSCs with ZA also produced a 50% reduction in the levels of secretion of IL-6 that has been previously shown to act as a potent paracrine growth factor for human breast cancer cells. Both RANTES and IL-6 transcripts were significantly reduced in MSCs following exposure to ZA, suggesting a transcriptional regulation of these cytokines by ZA. Conditioned medium from ZA-treated MSCs showed a reduced ability to promote the migration of ER-positive MCF-7 breast cancer cells through a fibronectin-coated membrane as compared with conditioned medium from untreated cells. In addition, antibodies anti-RANTES and anti-IL-6, alone or in combination, produced an approximately 50% reduction of the migration of MCF-7 induced by conditioned medium from untreated MSCs. In co-culture assays, treatment with ZA reduced the ability of MSCs to sustain the growth of breast cancer cells. Finally, the migration of MSCs was reduced by approximately 45% following treatment with ZA. Taken together, these data suggest that ZA might exert its antitumor activity in the bone marrow microenvironment by inhibiting the migration of MSCs and by blocking the ability of MSCs to secrete factors involved in breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 565.

Effect of zoledronic acid on oral fibroblasts and epithelial cells: a potential mechanism of bisphosphonate‐associated osteonecrosis

Osteonecrosis of the jaw secondary to bisphosphonate infusion (zoledronic acid-ZA) is assumed to be a bone disease. This study investigated the effects of ZA on soft tissues using oral mucosal cells as an in vitro model of soft tissue cell death in the pathogenesis of bone necrosis. Human gingival fibroblast and keratinocyte cell lines were exposed to different concentrations of ZA (0.25-3 micromol/l), using 1 micromol/l as the expected baseline concentration. A dose-response effect on apoptosis and cell proliferation [Terminal deoxynucleotidyl transferase-mediated dUTP-Biotin End Labelling and Annexin V or Coulter counter and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), respectively] was observed with increasing ZA concentrations; both reversed using siRNA against caspase 3 or 9. Gene expression analysis using RT(2) Profiler polymerase chain reaction Arrays demonstrated the differential expression of multiple genes involved in apoptosis including those that encode TNF, BCL-2, Caspase, IAP, TRAF and Death Domain families. Western blot analysis confirmed the presence of activated forms of caspase 3 and 9 and underexpression of survivin protein expression. This study demonstrated that low concentrations of ZA rapidly and directly affected the oral mucosal tissues though the induction of a gene-regulated apoptotic process. These findings support the potential for soft tissue injury as an initiating/potentiating event for osteonecrosis.

Zoledronic Acid Inhibits the Function of Toll-Like Receptor 4 Ligand Activated Dendritic Cells.

Zoledronic acid (ZA; Zometa®) is a cyclic nitrogen-containing bisphosphonate with anti-tumor activity used to treat patients with malignant diseases. As a side effect, ZA induces inflammatory responses which are accompanied by expansion of gd T cells that probably mediate its anti-tumor activity. In our study we analyzed the effects of ZA on the function and differentiation of monocyte-derived dendritic cells (DC). We investigated the differentiation of immature and LPS-stimulated DCs incubated with different ZA concentrations which are usually achieved in treated patients. Expression of DC-specific marker molecules was assessed by FACS. Furthermore, the activation state and involvement of different signal transduction pathways were evaluated by Western blot. To monitor DC function we used a migration assay and analyzed the cytokine secretion. We found that DC activation with TLR4 ligand LPS is modulated by ZA. We show that the expression of the surface maturation markers CD80, CD86, CD40, CD54, CD83 and DC-SIGN is diminished with increasing ZA levels upon LPS activation. The migratory capacity of LPS stimulated DCs towards the chemokine CCL19/MIP-3beta and the secretion of the activatory cytokine IL-12 are reduced at higher ZA levels. Increasing ZA concentrations lead to the down-regulation MyD88 and diminish the nuclear localization of NF-kB family members RelB and c-Rel as well as interferon-regulatory factors IRF-3 and IRF-8 in LPS activated DCs. Hence, ZA concentrations used to treat patients have inhibitory effects on DC differentiation and activation. This might lead to immuno-suppression and negatively affect tumor rejection or result in infectious complications.

Influence of Sodium Aluminosilicate, Hydroxy-Sodalite, Carnegieite, Aluminum Sulfate, and Aluminum Phosphate on Performance of Commercial Leghorns

Experiments were conducted to determine if the high ion-exchange capacity of sodium aluminosilicate (ZA) marketed as ETHACAL™ Feed Component or its aluminum content or both are related to the beneficial effect of ZA on egg specific gravity (ESG). In Experiments 1 and 2, ZA was compared with hydroxy-sodalite (HS) and carnegieite, which have the same chemical formula as ZA but little or no ion-exchange capacity. Two levels of ZA (0 and 1.5% of the diet) and three levels of HS (0, .75, and 1.5%) were fed in Experiment 1. In Experiment 2, 0, .75, and 1.5% of ZA and carnegieite were fed. In Experiment 3, ZA, carnegieite, aluminum sulfate, and aluminum phosphate were fed at levels calculated to contain .148 and .101% aluminum. Criteria evaluated were ESG, egg production, feed consumption, and egg weight.Egg specific gravity was significantly improved by ZA in all experiments and was not affected by HS, carnegieite (Experiments 1 and 2), aluminum sulfate (.148% Al), or aluminum phosphate (Experiment 3). Carnegieite and aluminum (.101%) from aluminum sulfate increased ESG in Experiment 3. In Experiments 1 and 3, egg production and feed consumption were not influenced by various treatments. Carnegieite and ZA reduced egg production and feed consumption in Experiment 2. It was concluded that the ion-exchange capacity and aluminum content of ZA may contribute to its beneficial effect on ESG.

Cyclic conjugation and the hückel molecular orbital model

A new graphic polynomial, μ (G, t), has been introduced which depends on a vectort and which reduces to the characteristic and matching polynomial of the graphG for certain choices oft. This leads to a unification of a great part of the previously developed theories of the characteristic and matching polynomials. The basic properties of μ (G, t) are determined. A method has been developed by which a functionJ (t) can be associated with every π-electron indexJ. Since the componentt a of the vectort is interpreted as the extent by which a particular cycleZ a of the molecular graphG influences the polynomial μ(G, t), it is possible to use the functionJ(t) in the study of the effect ofZ a on the π-electron indexJ. Total π-electron energy, π-electron charge and π-electron bond order have been analysed by this method and a number of topological rules of the modulo 4 type have been formulated. It is indicated that all these rules have a common algebraic background.