Harry S. Sitren

Total Parenteral Nutrition in the Mouse: Development of a Technique

A method for total parenteral nutrition in the mouse was developed using commercially available supplies and equipment. The mouses inferior vena cava was catheterized and the catheter was exteriorized from the tail. Mice (average body weight 22.5 g) were not tethered but instead were partially restrained by immobilizing the tail to protect the infusion tubing. A solution was formulated to contain 40% dextrose and 4.3% amino acids plus vitamins, electrolytes, and trace elements. It was administered via pump in graded fashion for the first 3 days to allow the mice to adapt, and then at a rate of 8 ml/day thereafter. This volume provided approximately 12 kcal and 54 mg nitrogen per day and was calculated to meet the needs of the mouse fed per os according to guidelines of the National Research Council. During the adaptation period mice lost weight but they were in positive N balance thereafter. At the end of 12 days body weight was not different than at the start, indicating the adequacy of nutrient input. Further, there were no differences in nitrogen balance or body weight in total parenteral nutrition fed mice in comparison with tail-restrained mice given the same solution per os. All mice appeared to tolerate the partial restraint without incidence and showed no untoward side effects. The rationale and validity of this technique is discussed in detail.

The zinc transporter Zip14 influences c-Met phosphorylation and hepatocyte proliferation during liver regeneration in mice.

BACKGROUND & AIMS Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes. METHODS Wild-type and Zip14(-/-) mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal-regulated kinase 1/2 signaling. RESULTS Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14(-/-) mice. CONCLUSIONS The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease.

Further studies of a technique for total parenteral nutrition in the mouse

Abstract A method for total parenteral feeding in mice has undergone further evaluation in our laboratory. The procedure involved cannulation of the inferior vena cava, partial immobilization by tail restraint and continuous infusion of a solution containing 40% dextrose plus 4.3% amino acids. In previous studies with this method (1,14), no evidence of restraint stress was noted, although adrenal function was not assessed. This report demonstrates that, in tail-restrained mice, urinary excretion of corticosteroids and catecholamines is not increased, thus indicating that this form of immobilization does not stimulate the hypothalamo-adrenocortical system. The technique of parenteral feeding was further evaluated in a group of female Swiss mice that were fully supported by parenteral infusions at maintenance levels for seven days. No changes in body weight were found. The weights of the spleen, kidneys, lungs and heart were normal when compared with controls. Liver weight declined, although protein and fat concentrations were normal. Carcass water, fat and nitrogen content were also normal. Hepatocellular enzyme activities were slightly altered. However, a group of mice given the parenteral solution to drink showed similar changes in enzyme activities, indicating that diet composition and not route of feeding was the responsible factor. Plasma protein and albumin levels were normal in parenterally-fed mice, whereas glucose decreased 28% with no detectable rise in insulin level. No evidence of immobilization-induced stress was found. Mice infused at NAS/NRC (2) levels of energy and nitrogen appeared to show normal utilization of the nutrients. A comparison of these results with those reported in parenterally-fed rats indicates that mice show fewer metabolic and compositional alterations associated with parenteral nutrition. This study further validates our technique of parenteral feeding in the mouse.

Inhibition of tumor growth and metastasis by chronic intravenous infusion of prostaglandin E1.

The role of prostaglandins and their synthesis inhibitors in malignant disease is undefined. The following studies were done to determine the effects of continuous intravenous prostaglandin E1 (PGE1) or a prostaglandin synthesis inhibitor, indomethacin, on tumor growth and metastasis in mice bearing Lewis lung carcinoma. Male B6D2F1 mice underwent tumor implantation in the right axilla on day 0. After 10 days of tumor growth, mice underwent intravenous (IV) catheterization and were infused with either PGE1 at 3 μg/kg/minute (PG-LOW), PGE1 at 6 μg/kg/minute (PG-HIGH), indomethacin (INDO) at 1 μg/kg/minute, or normal saline (NS). After 10 days of infusion, tumor volume, tumor weight, and the number of metastases greater than 2 mm in diameter were significantly decreased, and tumor doubling time was significantly prolonged in the PG-HIGH group compared to NS controls. None of the other experimental groups showed differences in these parameters. A second experiment with a similar experimental design was done infusing PGE, at 6 μg/kg/minute and at 12 μg/kg/minute to determine the maximum dose response of IV PGE1. Again a decrease in tumor volume, tumor weight, and metastatic rates were identified when compared to saline control, but there were no significant difference between the two doses of PGE1.

Correlation of estrogen, progesterone, and androgen receptors in breast cancer.

Breast cancer was induced in female Holtzman rats by intragastric administration of 7,12-dimethylbenz[a]antracene (DMBA). At tumor maturity, biopsies of viable tissue were obtained, frozen, and then assayed for estrogen, progesterone, and androgen receptor content. By simple linear regression analysis, progesterone receptor levels significantly correlated with both estrogen and androgen receptor levels, whereas estrogen and androgen receptor levels did not correlate with each other. Multiple regression analyses further substantiated the predictive value of the progesterone receptor for the other two hormone receptors. Knowledge of breast tumor androgen receptor levels may further enhance the value of the estrogen receptor and progesterone receptor in hormonal responsiveness. Further, the progesterone receptor may be the most sensitive of the steroid hormone receptors for selecting patients likely to respond to hormonal therapy.

Cost Containment Using Cysteine HCl Acidification to Increase Calcium/ Phosphate Solubility in Hyperalimentation Solutions

The purpose of this study was to determine if (1) the calcium/phosphate insoluble product was inversely related to pH [when cysteine HC1 (CH) was added as neonatal supplementation at 0.5 mM/kg/day to hyperalimentation (HAL) solutions] and (2) the potential cost savings to the hospital. The pH of the HAL solutions was adjusted by adding various amounts of CH to the HAL solution. HAL solutions containing 27 mEq of calcium/liter and 30 mEq (15 mM) of phosphate/liter were compounded. Ten-milliliter aliquots were analyzed at 0, 12, 24, and 48 hr. All samples (n = 56) were filtered (0.22 mu), viewed with 7-10,000 X magnification scanning electron microscopy, and qualitatively analyzed with a Philips Energy Dispersive X-Ray Analysis System equipped with a SW9100 Microprocessor. Calcium/phosphate insoluble product was present in the 0-, 12-, 24-, and 48-hr samples from the CH-free solutions. The solutions containing 759 mg (4.17 mM)/liter of CH however, remained free of precipitant. This investigation demonstrated that addition of CH to HAL can foster significant cost containment (projected

Changes in Circadian Rhythmicity of Liver and Serum Parameters in Rats Fed a Total Parenteral Nutrition Solution by Continuous and Discontinuous Intravenous or Intragastric Infusion

82,000/yr tangible hospital savings) by the elimination of current calcium/phosphate separation procedures for neonates on parenteral nutrition.

The effect of energy substrate manipulation on tumor growth and metastasis and intermediary metabolism in the parenterally fed mouse

Circadian rhythms were assessed in rats (224-246 grams) receiving either an intragastric (IG) or intravenous (IV) infusion continuously (C), or IG or IV infusions discontinuously (D) from 0000 to 1400 hours daily. A control group was maintained on a stock diet ad libitum. IV- and IG-fed rats were infused with 50 milliliters per day of a solution containing 25% dextrose and 4.25% amino acids plus vitamins and minerals. After 10 to 11 days, groups of rats were killed from each treatment every 6 hours over a 24-hour period. Results showed that IG feeding promoted better growth and nitrogen retention than IV feeding. Rhythm patterns for liver weight, glycogen, and protein content were similar between D-IG and D-IV infused rats. All groups except the D-IV fed rats exhibited fluctuations of serum insulin. A rhythm was not present for serum glucose in C-IV fed rats and a rhythm was also absent for serum albumin in both C-IV and D-IV fed rats. C-IV feeding increased serum glucose and insulin levels and decreased serum albumin levels in comparison to IG feeding. In comparison to the ad libitum-fed, control rats, C-IV or D-IV or IG infusions led to alterations in liver and serum parameters.

Total Parenteral Nutrition in the Mouse: Body Composition and Plasma Chemistries

The effect of N-free energy substrate manipulation on tumor growth and metastasis, host maintenance, and intermediary metabolism was studied in parenterally fed Swiss mice bearing subcutaneously implanted Lewis lung carcinoma. Non-N energy was provided from dextrose (CHO), lipid emulsion (FAT), or a 75:25 balanced (BAL) solution, infused from day 14 through day 22 postimplant. Control mice were offered equivalent energy and N from a balanced, casein-based solid diet (CAS). Tumor-doubling time was significantly prolonged in the CHO group compared to FAT and CAS. Pulmonary metastatic nodules were decreased in number in all parenterally fed mice compared to CAS, suggesting that the route of administration altered pulmonary physiology in such a way that the transmissability and/or growth of the tumor cells was inhibited. Tumor-free body weight was maintained in the CHO (+ 1.3%) and BAL (+ 0.3%) groups. However, significant weight loss occurred, despite equal intake, in the FAT (-4.7%) and CAS (-7.5%) groups. The energy appeared to be channeled into nonoxidative pathways, reflected by an increase in hepatic and adipose tissue lipogenesis and hepatic glycogen content. During the period studied, parenteral dextrose/amino acid infusion in this host-tumor system resulted in a decrease in primary tumor growth and optimal host maintenance compared to fat-based TPN and enteral feeding of a balanced, solid diet. Tumor metastasis was decreased in all parenterally fed mice, a phenomenon related to the route of administration and apparently independent of energy substrate.

Nutritional Support by Intraperitoneal Dialysis in the Rat: Maintenance of Body Weight with Normal Liver and Plasma Chemistries

Mice that were maintained in energy and nitrogen (N) balance by total parenteral nutrition (TPN) for 12 days were analyzed for changes in organ weight, carcass and liver N and fat, and plasma glucose, urea N, and total protein. The results are compared with two other groups: (1) PO, which consisted of mice that were given the TPN solution per os in amounts equivalent to the TPN group, and (2) AL, which consisted of mice allowed to consume a stock diet ad libitum. In comparison with group AL, the TPN-fed mice had normal liver, kidney, and lung weights but heavier spleens and hearts. Group PO had an increase in liver weight only. Hepatic lipid content declined in group TPN but increased markedly in PO-fed mice. The latter group also demonstrated a 35% increase in carcass fat whereas it was unchanged in the TPN group. No differences were found in plasma urea N and total protein among the groups but plasma glucose increased 2-fold in group PO. It appears that our technique of parenteral feeding in mice maintains fairly normal body composition and plasma chemistries. However, mice drinking the TPN solution (group PO) exhibited the greatest number of alterations. These results are discussed in relation to differences in route of feeding, diet composition, feeding pattern, and the possible influence of circadian rhythms. The dilemma of choosing appropriate control groups in TPN studies is also discussed.