In vitro and in silico assessment of antitumor properties and biomolecular binding studies for two new complexes based on NiII bearing k2N,S-donor ligands.

This work deals with two new molecule-based materials, namely NiII-complexes of general formulae [Ni(L1)2] (Ni1) and [Ni(L2)2] (Ni2), where L1 = trans-cinnamaldehyde-N(4)-methyl thiosemicarbazone and L2 = trans-cinnamaldehyde-N(4)-ethyl thiosemicarbazone, as potential antitumor agents. Both compounds were characterized by elemental analysis, molar conductivity and spectroscopic techniques (FTIR and NMR). Their molecular structures were obtained by single-crystal X-ray diffraction analysis. Each one crystallizes in a monoclinic space group P 21/c, also the asymmetric unit comprises of one NiII ion located on an inversion centre and one anionic ligand, which acts as a κ2N,S-donor affording a five-membered metallaring. The compounds were screened against two selected tumour cell lines (MCF-7 and A549) and non-tumour fibroblasts cell line (MRC-5) via MTT assays. In both tumour cells, all compounds exhibited higher cytotoxicity than the control drug (cisplatin). The IC50 values ranges of 3.70 - 41.37 µM and 1.06 - 14.91 µM were found for MCF-7 and A549, respectively. Importantly, all of them were less toxicity than cisplatin in MRC-5 with SI values ranged at 11.80 - 86.60. The red blood cell (RBC) assay revealed Ni2 as non-toxic due to its reduced haemolytic effect (0--9% at 1--10 µM). The DNA binding was investigated through a combination of spectrophotometric absorption and emission titrations, electrophoresis, and circular dichroism experiments. As a result, these metal complexes were not able to strongly binding to DNA (Kb values ~104 mol L--1) but suggesting groove-binding interactions. The scavenging ability of them towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical was also evaluated in this work, but no important antioxidant behaviour was detected. Further, the interaction of Ni1 and Ni2 to human serum albumin (HSA) was explored by quenching of tryptophan emission, warfarin competitive assay, and molecular docking protocols. The HSA binding analyses indicated good affinity of both complexes to Sudlow site I (Kb values ⁓103 mol L-1).

Variations in maternal adenylate cyclase genes are associated with congenital Zika syndrome in a cohort from Northeast, Brazil.

BACKGROUND: Vertical transmission of Zika virus (ZIKV) is associated with congenital malformations but the mechanism of pathogenesis remains unclear. Although host genetics appear to play a role, no genetic association study has yet been performed to evaluate this question. In order to investigate if maternal genetic variation is associated with Congenital Zika Syndrome (CZS), we conducted a case-control study in a cohort of Brazilian women infected with ZIKV during pregnancy. METHODS: A total of 100 women who reported symptoms of zika during pregnancy were enrolled and tested for ZIKV. Among 52 women positive for ZIKV infection, 28 were classified as cases and 24 as controls based on the presence or absence of CZS in their infants. Variations in the coding region of 205 candidate genes involved in cAMP signaling or immune response were assessed by high throughput sequencing and tested for association with development of CZS. RESULTS: From the 817 single nucleotide variations (SNVs) included in association analyses, 22 SNVs in 17 genes were associated with CZS under an additive model (alpha = 0.05). Variations c.319T>C (rs11676272) and c.1297G>A, located at ADCY3 and ADCY7 genes showed the most prominent effect. The association of ADCY3 and ADCY7 genes was confirmed using a Sequence Kernel Association Test to assess the joint effect of common and rare variations, and results were statistically significant after adjustment for multiple comparisons (P < 0.002). CONCLUSION: These results suggest that maternal ADCY genes contribute to ZIKV pathogenicity and influence the outcome of CZS, being promising candidates for further replication studies and functional analysis.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea.

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Interleukin-6 levels in the conjunctival epithelium of patients with dry eye disease treated with cyclosporine ophthalmic emulsion.

PURPOSE: To evaluate interleukin-6 (IL-6) levels in the conjunctival epithelium of patients with moderate to severe dry eye disease before and after treatment with cyclosporin A ophthalmic emulsion (CsA) or its vehicle. METHODS: Conjunctival cytology specimens were obtained from a subset of patients enrolled in a 6-month randomized, double-masked clinical trial of the efficacy and safety of topical CsA at baseline and after 3 and 6 months of B.I.D. treatment with 0.05% cyclosporine emulsion (n = 13), 0.1% cyclosporine emulsion (n = 8), or vehicle (n = 10). RNA was extracted and a competitive reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the levels of mRNA encoding the inflammatory cytokine IL-6 and a housekeeping gene, G3PDH. Levels of IL-6 and G3PDH were measured and compared. RESULTS: There was no change from baseline in the level of G3PDH after 3 or 6 months in any group. IL-6 normalized for G3PDH (IL-6/G3PDH ratio) was not different from baseline at 3 months but showed a significant decrease from baseline in the group treated with 0.05% CsA (p = 0.048) at 6 months. No significant between-group differences were noted and no correlation was observed between the change in IL-6/G3PDH and corneal fluorescein staining. CONCLUSIONS: This preliminary, small-cohort study showed a decrease in IL-6 in the conjunctival epithelium of moderate to severe dry eye patients treated with 0.05% CsA for 6 months. The observed decrease suggests that dry eye disease involves immune-mediated inflammatory processes that may be decreased by treatment with topical ophthalmic cyclosporine.

Reliability and validity of the Ocular Surface Disease Index.

OBJECTIVE: To evaluate the validity and reliability of the Ocular Surface Disease Index (OSDI) questionnaire. METHODS: Participants (109 patients with dry eye and 30 normal controls) completed the OSDI, the National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25), the McMonnies Dry Eye Questionnaire, the Short Form-12 (SF-12) Health Status Questionnaire, and an ophthalmic examination including Schirmer tests, tear breakup time, and fluorescein and lissamine green staining. RESULTS: Factor analysis identified 3 subscales of the OSDI: vision-related function, ocular symptoms, and environmental triggers. Reliability (measured by Cronbach alpha) ranged from good to excellent for the overall instrument and each subscale, and test-retest reliability was good to excellent. The OSDI was valid, effectively discriminating between normal, mild to moderate, and severe dry eye disease as defined by both physician's assessment and a composite disease severity score. The OSDI also correlated significantly with the McMonnies questionnaire, the National Eye Institute Visual Functioning Questionnaire, the physical component summary score of the Short Form-12, patient perception of symptoms, and artificial tear usage. CONCLUSIONS: The OSDI is a valid and reliable instrument for measuring the severity of dry eye disease, and it possesses the necessary psychometric properties to be used as an end point in clinical trials.

Efficacy and safety of cyclosporin A ophthalmic emulsion in the treatment of moderate-to-severe dry eye disease: a dose-ranging, randomized trial. The Cyclosporin A Phase 2 Study Group.

OBJECTIVE: To investigate the efficacy, safety, formulation tolerability, and optimal dosing of a novel cyclosporin A oil-in-water emulsion formulation for the treatment of moderate-to-severe dry eye disease. DESIGN: Randomized, multicenter, double-masked, parallel-group, dose-response controlled trial. PARTICIPANTS: Total enrollment: 162 patients; cyclosporin A groups: 129 patients; vehicle group: 33 patients. INTERVENTION: Patients instilled study medication (cyclosporin A ophthalmic emulsion 0.05%, 0.1%, 0.2%, or 0.4%, or vehicle) twice daily into both eyes for 12 weeks, followed by a 4-week posttreatment observation period. EFFICACY: rose bengal staining, superficial punctate keratitis, Schirmer tear test, symptoms of ocular discomfort, and the Ocular Surface Disease Index (OSDI; a measure of symptom frequency and impact on vision-related functioning). SAFETY: biomicroscopy, cyclosporin A blood levels, conjunctival microbiology, intraocular pressure, visual acuity, and monitoring of adverse events. RESULTS: In a subset of 90 patients with moderate-to-severe keratoconjunctivitis sicca, the most significant improvements with cyclosporin A treatment were in rose bengal staining, superficial punctate keratitis, sandy or gritty feeling, dryness, and itching, with improvements persisting into the posttreatment period in some treatment groups. There was also a decrease in OSDI scores, indicating a decrease in the effect of ocular symptoms on patients' daily lives. There was no clear dose-response relationship, but cyclosporin A 0.1% produced the most consistent improvement in objective and subjective end points and cyclosporin A 0.05% gave the most consistent improvement in patient symptoms. The vehicle also performed well, perhaps because of its long residence time on the ocular surface. There were no significant adverse effects, no microbial overgrowth, and no increased risk of ocular infection in any treatment group. The highest cyclosporin A blood concentration detected was 0.16 ng/ml. All treatments were well tolerated by patients. CONCLUSIONS: Cyclosporin A ophthalmic emulsions, 0.05%, 0.1%, 0.2%, and 0.4%, were safe and well tolerated, significantly improved the ocular signs and symptoms of moderate-to-severe dry eye disease, and decreased the effect of the disease on vision-related functioning. Cyclosporin A 0.05% and 0.1% were deemed the most appropriate formulations for future clinical studies because no additional benefits were observed with the higher concentrations.

Two multicenter, randomized studies of the efficacy and safety of cyclosporine ophthalmic emulsion in moderate to severe dry eye disease. CsA Phase 3 Study Group.

OBJECTIVE: To compare the efficacy and safety of cyclosporin A ([CsA] 0.05% and 0.1% ophthalmic emulsions) to vehicle in patients with moderate to severe dry eye disease. DESIGN: Multicenter, randomized, double-masked, parallel-group, 6-month, vehicle-controlled. PARTICIPANTS: A total of 877 patients with defined moderate to severe dry eye disease (292 to 293 in each treatment group). METHODS: Two identical clinical trials; patients were treated twice daily with either CsA, 0.05% or 0.1%, or vehicle. The results of these two trials were combined for analysis. EFFICACY: corneal and interpalpebral dye staining, Schirmer tear test (with and without anesthesia), tear break-up time, Ocular Surface Disease Index (OSDI), facial expression, patient subjective rating scale, symptoms of dry eye, investigator's evaluation of global response to treatment, treatment success, and daily use of artificial tears. SAFETY: occurrence of adverse events, best-corrected visual acuity, intraocular pressure, biomicroscopy, and blood trough CsA concentrations. RESULTS: Treatment with CsA, 0.05% or 0.1%, gave significantly (P < or = 0.05) greater improvements than vehicle in two objective signs of dry eye disease (corneal staining and categorized Schirmer values). CsA 0.05% treatment also gave significantly greater improvements (P < 0.05) in three subjective measures of dry eye disease (blurred vision, need for concomitant artificial tears, and the physician's evaluation of global response to treatment). There was no dose-response effect. Both CsA treatments exhibited an excellent safety profile, and there were no significant topical or systemic adverse safety findings. CONCLUSIONS: The novel ophthalmic formulations CsA 0.05% and 0.1% were safe and effective in the treatment of moderate to severe dry eye disease yielding improvements in both objective and subjective measures. Topical CsA represents a new pharmacologically based treatment for dry eye disease that may provide significant patient benefits.

Evaluation of subjective assessments and objective diagnostic tests for diagnosing tear-film disorders known to cause ocular irritation.

PURPOSE: To determine which subjective assessments and objective tests have clinical utility as diagnostic tools in ocular irritation associated with Sjögren's syndrome-related aqueous tear deficiency (ATD), non-Sjögren ATD, inflammatory meibomian gland disease (MGD) associated with rosacea, and atrophic MGD. METHODS: Forty adults with ocular irritation and 10 with normal ocular surfaces were enrolled in a nonrandomized, nonblinded clinical trial. Symptoms were evaluated. Tests included biomicroscopy; evaluation of tear-film integrity, production, and clearance; fluorescein and rose bengal staining; and serum autoantibody screening. RESULTS: Symptoms were similar among groups and most severe in the Sjögren's group. Fluorescein tear break-up time was significantly faster in the ATD and MGD groups than that in controls. Schirmer scores were significantly lower in the ATD group than those in MGD and control groups. Tear clearance was delayed in the ATD and atrophic MGD groups. Xeroscope grid distortion was noted only with ATD. The Sjögren's group had greater loss of naso-lacrimal reflex, slower fluorescein clearance, and greater ocular-surface fluorescein and rose bengal staining than did the others. More MGD subjects had meibomian gland orifice metaplasia and acinar dropout than did those with Sjögren-related ATD and controls. Schirmer scores correlated inversely with rose bengal staining, corneal fluorescein staining, and grid distortion. Rose bengal staining correlated with grid distortion and loss of nasal-lacrimal reflex, but not with MGD. CONCLUSION: Subjective assessments and objective diagnostic tests have clinical utility as diagnostic tools in tear-film disorders. ATD is correlated with ocular-surface disease. An algorithm summarizing the diagnostic utility of these tests is included.

Correlation of goblet cell density and mucosal epithelial membrane mucin expression with rose bengal staining in patients with ocular irritation.

PURPOSE: This study was designed to compare goblet cell densities and mucosal epithelial membrane mucin (MEM) expression in impression cytology specimens obtained from control subjects and patients with one of the following clinically defined diseases: aqueous tear deficiency (ATD) associated with Sjögren syndrome, ATD not associated with Sjögren syndrome, inflammatory Meibomian gland disease associated with rosacea, and Meibomian gland atrophy. These data were correlated with ocular surface rose Bengal staining scores, Schirmer scores, and HLA-DR antigen staining of conjunctival epithelial cells. METHODS: Goblet cell density and MEM expression were studied by impression imprints with immunohistochemical staining using an anti-mucosal epithelial membrane mucin antibody in the temporal and inferior bulbar and inferior tarsal conjunctiva of study subjects. RESULTS: Goblet cell density adjacent to the temporal limbus was significantly reduced at 3 mm posterior to the temporal limbus in both aqueous tear deficiency groups compared with the other groups and in patients with Sjögren syndrome compared with all other groups. In the inferior tarsus, goblet cell density was significantly reduced in patients with non-Sjögren syndrome ATD as compared with all other groups, except those with inflammatory Meibomian gland disease. Mucosal epithelial membrane mucin expression in the bulbar and tarsal conjunctiva was absent in a greater percentage of patients with Sjögren syndrome compared with all other groups. Total ocular surface rose Bengal staining scores were significantly higher in patients with Sjögren syndrome as compared with all other groups and in patients with non-Sjögren syndrome ATD as compared with control groups. Rose Bengal staining scores and Schirmer I test results (without anesthesia) were inversely correlated with bulbar, but not tarsal, conjunctival goblet cell densities, and with the absence of bulbar conjunctival MEM expression. CONCLUSIONS: These results suggest that reduced goblet cell density and mucosal epithelial cell mucin expression could explain increased rose Bengal staining in patients with aqueous tear deficiency. In addition, MEM may be regarded as a marker for normal differentiation of ocular surface epithelia, with its absence signifying the development of squamous metaplasia.

Effects of somatotropin and substrates on patterns of liver metabolism in lactating dairy cattle.

Objectives of this study were to quantitate metabolite fluxes in ruminant liver and to delineate effects of recombinant bST on patterns of nutrient metabolism by liver. Nineteen multiparous cows ranging in previous lactational performance from 6400 to 13,500 kg per 305-d lactation were treated with either placebo or bST (40 mg/d) from wk 11 to 18 of lactation. Liver tissue was collected at slaughter. Tissue slices were incubated with various 14C-labeled substrates, and rates of conversion of label to CO2 and metabolites were measured. In vivo recombinant bST treatment increased in vitro conversion of [1-14C]propionate and [2-14C]acetate to glucose more than twofold. At 2.5 mM propionate, bST-treated cows converted propionate to glucose at 90% efficiency. Recombinant bST increased [14C]bicarbonate incorporation into glucose five-fold. Overall, bST treatment resulted in greater C flow from propionate and acetate through the TCA cycle. Acetate had only small effects on propionate metabolism and no effects on lactate plus pyruvate metabolism. Unexpectedly, propionate decreased acetate conversion to ketone bodies. Suggested mechanisms for this observation include depletion of coenzyme A and allosteric regulation of carnitine palmitoyltransferase I by methylmalonyl-coenzyme A formed from propionate. In summary, bST treatment resulted in increased rates of gluconeogenesis and oxidation in liver in support of lactation.

Kinetics of amino acid extraction by lactating mammary glands in control and sometribove-treated Holstein cows.

Studies of mammary arteriovenous difference were conducted on multiparous lactating Holstein cows (n = 21) on d 35, 70, 105, and 126 of lactation to examine kinetic relationships between arterial concentration and mammary gland extraction of AA. Additionally, these cows were paired by previous lactational performance and assigned to bST-treated or control groups to examine the effect of bST treatment on AA concentration and extraction by lactating mammary glands. Treated cows were injected daily with 40 mg of recombinant bST from d 71 through 126 of lactation. Arterial concentrations of Asp, Ser, Asn, Gly, beta-aminoisobutyrate, and Met were increased. Concentrations of Val, Ile, Leu, Phe, Orn, and Lys were decreased in bST-treated cows compared with controls. Increased extractions of Asp and Met by mammary glands in treated versus control cows were correlated positively with treatment-induced changes in arterial concentrations of these AA. However, increased mammary extractions of Arg, cystathionine, Leu, and Lys by bST-treated compared with control cows were not correlated with bST-induced changes in arterial concentrations of these AA. Extractions of Asn, His, Thr, Arg, Tyr, Met, cystathionine, cystine, Ile, Phe, Orn, Glu, Gly, Tau, Cit, Leu, and Val were correlated linearly with arterial concentrations (r2 greater than .15) of each AA. Extractions of Asp, Glu, Ser, Asn, Gly, Gln, Tau, His, Cit, Thr, Pro, Tyr, Val, cystine, Ile, Leu, Trp, Orn, and Lys also were correlated with arteriovenous differences of Met.

Patterns of nutrient uptake by the mammary glands of lactating dairy cows.

Twenty-one multiparous lactating dairy cows with previous 305-d milk production records varying from 5900 to 13,600 kg were used to examine patterns of nutrient uptake by the mammary glands. On d 71 and continuing until d 126 of lactation, animals were injected daily with 40 mg of sometribove (bST group) or bicarbonate buffer (control group). Arterial and venous blood plasma samples were collected over a 12-h period on d 35, 70, 105, and 126 of lactation. Regression equations developed to evaluate linear effects of plasma arterial concentrations on net arterial-venous difference across the mammary glands demonstrated that, for acetate, NEFA, and D-beta-hydroxybutyrate, plasma arterial concentration accounted for over 50% of variation in uptake by the mammary glands. Additionally, a sigmoidal equation fitted the relationship between D-beta-hydroxybutyrate plasma arterial concentration and mammary gland uptake (r2 = .70). Triacylglyceride concentration was less effective in predicting uptake (r2 = .25). Administration of bST did not alter patterns of nutrient uptake, but a fourfold increase in NEFA uptake was predicted for bST-treated cows from this study, using NEFA concentrations from the literature. These observations indicate that plasma concentrations of acetate, NEFA, D-beta-hydroxybutyrate, and triacylglyceride are major determinants of uptake by the mammary glands. Factors other than plasma concentration, such as mammary gland biosynthetic capacity, availability of other nutrients, and blood flow, determine uptakes of glucose, lactate, and total and free cholesterol (r2 less than or equal to .03).

Relationship of early lactation and bovine somatotropin on nutrient uptake by cow mammary glands.

Twenty-one multiparous lactating dairy cows with previous 305-d milk production records varying from 5900 to 13,600 kg were used to investigate effects of bST administration and stage of lactation on nutrient plasma arterial concentrations and arterial-venous differences across the mammary glands (uptake). On d 71 and continuing until d 126 of lactation, cows were injected with 40 mg of sometribove (bST group) or bicarbonate buffer (placebo group). Arterial and venous blood plasma samples were collected over a 12-h period on d 35, 70, 105, and 126 of lactation. Plasma concentration of glucose was 7% higher in midlactation compared with early lactation cows. Plasma concentration of acetate decreased from 2.11 to 1.87 mM in placebo versus bST-treated cows. Plasma arterial concentration and uptake of D-beta-hydroxybutyrate were .52 and .18 mM higher, respectively, in early versus midlactation cows. Concentration and uptake of NEFA were elevated in both early lactation and bST-treated cows. Triacylglyceride concentrations were 24 and 19% lower in early lactation and bST-treated cows compared with midlactation cows receiving placebo. Likewise, uptake of triacylglyceride was reduced in early lactation and with bST treatment compared with midlactation. The mediation of nutrient delivery and uptake by the mammary gland appears to be markedly similar between early lactation and bST-treated cows, suggesting a similarity between these physiological states and the homeostatic and homeorhetic mechanisms regulating nutrient partitioning in the lactating dairy cow. In early compared with midlactation cows receiving placebo, uptakes of D-beta-hydroxybutyrate and NEFA were reduced, and triacylglyceride uptake increased.

Whole blood and plasma amino acid uptakes by lactating bovine mammary glands.

The objective of the experiment was to determine the contribution of red blood cells to transport of individual amino acids to lactating bovine mammary glands. Blood samples were collected from coccygeal and subcutaneous abdominal veins of 21 lactating Holstein cows on d 35, 70, 105, and 126 of lactation. Samples were collected every 20 min for 12 h. Subsamples of whole blood and plasma were pooled by hour and day. Hourly plasma samples and daily whole blood and plasma samples were analyzed for amino acid concentration. Plasma glutamate concentration was stable throughout the 12-h collection period, indicating that sample collection did not perturb amino acid homeostasis. Therefore, data from pooled daily samples were used for subsequent comparisons. Whole blood arteriovenous differences of phosphoserine, aspartate, glutamate, hydroxyproline, phosphoethanolamine, serine, asparagine, glycine, glutamine, taurine, histidine, citrulline, threonine, alanine, beta-aminoisobutyrate, carnosine, arginine, proline, alpha-aminobutyrate, tyrosine, valine, methionine, cystine, isoleucine, leucine, phenylalanine, tryptophan, ornithine, and lysine differed significantly from plasma arteriovenous differences. Uptakes of individual amino acids from plasma were poorly correlated with uptake from whole blood. These data clearly indicate that uptake data derived from plasma do not adequately represent whole blood amino acid uptake.

Longitudinal changes in the mineral composition of mouse milk and the relationship to zinc metabolism of the suckling neonate.

We studied changes in the mineral composition of milk of lactating Swiss-Webster mice and the relationship of those changes to mineral metabolism of suckling mouse pups. Concentrations of Zn, Cu, Mg and Ca were analyzed in maternal and neonatal tissues and in milk; Zn metabolism was studied using 65Zn. Although Cu, Ca and Mg concentrations in milk declined during the first 2 d of lactation, only the concentration of Zn decreased progressively throughout 30 d. Various pup tissues were characterized by developmental changes in concentrations of some elements. Turnover of Zn in neonatal tissues was studied by radiolabeling litters in utero and fostering them at birth to nonradiolabeled lactating dams. The turnover of whole-body 65Zn in suckling mice decreased during development, and, at 20 d of age, the biological half-life of 65Zn in the neonate was approximately the same as for a nonpregnant, nonlactating adult female (20 d). The decreased turnover of 65Zn in lactating dams that characterized progressive lactation and was reflected in the Zn concentration of milk is in agreement with changes in whole-body 65Zn turnover observed for the suckling mouse. These findings demonstrate that the metabolism of the suckling neonate is directly related to longitudinal changes in the composition of maternal milk.

Mineral status of mice suckling early-, mid- and late-lactating foster dams.

We have demonstrated that the zinc (Zn) concentration of mouse milk declines significantly over the lactation period. Pups radiolabeled in utero with 65Zn were forward-fostered (FF) to nonradiolabeled dams at a later stage of lactation to study the effects of early milk deprivation. Other groups of radiolabeled pups were back-fostered at 5, 10 and 15 days of age to a nonradiolabeled dam who had just given birth; this provided additional colostrum during the suckling period. Litters fostered at birth to an unlabeled dam at d 0 of lactation were used as controls. Weight gain of FF litters decreased and signs of Zn deficiency increased as the foster dam's days of lactation increased. The Zn concentration of kidney, brain and plasma tended to decrease with increasing lactation days of the foster dam. Tibia Zn concentration declined progressively as the lactation days of the foster dam increased, and the concentration of calcium also was lower in all three groups of FF litters than in controls, indicating that bone calcification may have been impaired. Kinetic data proved to be a more sensitive index of Zn status than tissue Zn concentration. The biological half-life of whole-body 65Zn for FF suckling mice increased in a linear fashion with increasing lactation days of the foster dam; whole-body retention of 65Zn for back-fostered litters did not differ from that for controls. Brain, small intestine, kidney muscle, plasma and tibia of pups FF to late-lactating dams seemed to have greater retention of Zn than did controls. Thus, deprivation of early milk impaired growth and development of the mouse neonate despite some ability to conserve Zn.

Mineral composition and zinc metabolism in female mice of varying age and reproductive status.

Zinc metabolism of adult female C57BL/6J mice varying in age and reproductive status was studied using 65Zn. Animals were injected intraperitoneally with isotope, and whole-body and tissue turnover of 65Zn was measured. Biological half-life of whole-body 65Zn for pregnant females was 129% of that for nonpregnant, nonlactating females of similar age (25.7 and 20.0 d, respectively). Conversely, the half-life of zinc was less for lactating (11.1 d) and aged (15.3 d) females than for either pregnant or nonpregnant, nonlactating young adult females. Retention of 65Zn was generally lower for all tissues of pregnant and lactating dams than for nonpregnant, nonlactating females except for brain and tibia. In aged females, specific activity at 20 d postinjection did not differ from that of nonpregnant, nonlactating young adult females for any tissue except bone. While specific activity of tibia increased during pregnancy and lactation, it was lower in aged females than in nonpregnant, nonlactating young adult females. Increased organ content of zinc resulted from changes in zinc concentration or in organ mass or both, and was supported by increased food intake. Thus, greater food intake for these groups than for young adult females contributed to the higher turnover of tissue 65Zn. Differences in concentrations of Cu, Ca and Mg were also observed among groups for some tissues; the significance of these differences and their relationship to zinc metabolism are not clear.

Effects of gastric nutritional supplementation on fetal umbilical uptake of nutrients.

Intrauterine nutritional supplementation may be of therapeutic benefit to the malnourished, growth-retarded fetus. Using 14 chronically catheterized, third trimester fetal lambs, we evaluated the effects of gastric infusions of amino acids and glucose on umbilical uptake of alpha-amino nitrogen, glucose, lactate, and oxygen. When amino acids were infused, amino nitrogen was gained through the intestine at an average rate equal to 45% of fetal umbilical uptake of amino nitrogen. There were no consistent changes in umbilical uptake of the measured nutrients, except for a small decrease in umbilical uptake of oxygen (P less than 0.05). No relationship were found between changes in fetal amino nitrogen levels or changes in fetal-maternal amino nitrogen concentration gradients and umbilical uptake of amino nitrogen. When glucose was infused, glucose was absorbed through the intestine at an average rate equal to 42% of fetal umbilical uptake of glucose. There were no changes in umbilical uptake of amino nitrogen, lactate, or oxygen. Umbilical uptake of glucose decreased, however, in inverse proportion to both the glucose infusion rate (P less than 0.005) and the rise in fetal glucose concentration (P less than 0.025). This limited the quantity of glucose that could be gained by the fetus, via gastrointestinal supplementation. During the infusions, we also observed a change in the pattern of fetal lower body blood flow. There was a 12% decrease in the mean umbilical blood flow (P less than 0.005) and a 22% increase in mean intestinal flow (P less than 0.05).

Consumption of carbohydrates, amino acids and oxygen across the intestinal circulation in the fetal sheep.

Since large volumes of nutrient rich amniotic fluid are swallowed by the fetus, it has been suggested that intestinal digestion and absorption contribute significantly to fetal nutrition. To see if nutrients are being gained across the intestine, we measured blood flow and intestinal arteriovenous concentration differences of glucose, alpha-amino nitrogen, lactate, fructose and oxygen in eleven third trimester fetal sheep with chronically implanted vascular catheters. We found that in fetal blood circulating through the intestine nutrient concentration decreased significantly with arterio-venous concentration differences for glucose of 0.78 +/- 0.21 (SEM) mg/dl (P < 0.002), for alpha-amino nitrogen of 0.52 +/- 0.15 mg/dl (P < 0.005), for lactate of 0.68 +/- 0.24 mg/dl (P < 0.05) and for oxygen of 1.50 +/- 0.08 ml/dl (P < 0.001). Fructose concentration did not change. Blood flow to the fetal intestine averaged 89.92 +/- 7.16 ml/min and the intestine consumed 0.74 +/- 0.24 mg of glucose, 0.43 +/- 0.17 mg of alpha-amino nitrogen, 0.83 +/- 0.28 mg of lactate and 1.37 +/- 0.14 ml of oxygen per minute. Compared to previously published values for the umbilical uptake of nutrients the fetal intestine metabolizes about 4% of the glucose, 6% of the alpha-amino nitrogen, 13% of the lactate and 6% of the oxygen obtained across the umbilical circulation. Intestinal absorption does not appear to serve as a source of simple nutrients for the rest of the fetus, in fact intestinal metabolism extracts significant amounts of nutrients from fetal blood.