Monte Carlo spreadsheet modeling of stable isotope biosynthesis.

Metabolic physiologists often introduce stable isotopes, atoms containing additional neutrons, into molecules during biosynthesis. This tags the newly synthesized material by altering its mass. Monte Carlo analysis is implemented on a popular spreadsheet to analyze this process. An example is provided where acetoacetate is synthesized by condensation of two acetate moieties. The precursor acetate is present as a mixture of natural, and 13C enriched, acetate. Monte Carlo spreadsheet modeling captures the complexity of the multi-species isotope biosynthesis by repetitively performing multiple simultaneous Boolean calculations. The effects of increasing the number of molecules synthesized on the goodness of fit of between model and an exact analytical solution is illustrated.

Isotopomer spectral analysis of cholesterol synthesis: applications in human hepatoma cells.

Cholesterol synthesis from 13C-labeled precursors produces a discrete spectrum of mass isotopomers detectable using gas chromatography-mass spectrometry. The isotopomer spectral analysis (ISA) method matches the observed spectrum of cholesterol isotopomers with a mathematical model to obtain the best fit of model spectrum to data spectrum. The model was based on multinomial probability expressions that simulate cholesterol synthesis as a condensation of mevalonate fragments. As many as four unknown parameters, representing fluxes between compartments, were included in the model. Models were developed to assess cholesterol synthesis from 13C-enriched precursors including mevalonate, acetate, acetoacetate or octanoate. Models were tested in the human hepatoma cell line, Hep G2, which readily incorporated the 13C substrates into cholesterol. The ISA approach was used to estimate the fractional amount of the cholesterol precursors derived from the 13C substrate and the fraction of total cellular cholesterol synthesized in the presence of the 13C substrate. The study demonstrated the feasibility of the ISA approach for a condensation biosynthesis that is not a simple polymerization and for models with more than two unknown parameters.

Verapamil enhances the survival of primary ischemic venous obstructed rodent skin flaps.

The effect of verapamil, a calcium channel blocker, on the survival of skin flaps subjected to primary venous obstruction was studied. Skin flaps 9 x 4 cm, which are axial patterns with random extension, were elevated in Sprague-Dawley rats. A microvascular clamp was placed on the vein alone for 8 hours. Group 1 received verapamil (0.3 mg/kg) before flap elevation and before clamp release; group 2 received saline on the same schedule. Group 3 received verapamil (0.3 mg/kg) as above, plus every 8 hours for 5 days postoperatively. Group 4 received saline on the same schedule. There was no difference in survival between groups 1 and 2. Group 3 had 100% improvement in the flap survival compared with group 4 (78% vs 37%). Verapamil, if administered for the duration of the experiment, significantly increased flap survival.

Isotopomer spectral analysis of triglyceride fatty acid synthesis in 3T3-L1 cells.

A new analysis of stable isotope data for biosynthesis reaction, isotopomer spectral analysis (ISA), is demonstrated. ISA is theoretically applicable for polymerization biosynthesis where data are collected using selected ion-monitoring gas chromatography-mass spectrometry. ISA utilizes the discrete spectrum of isotopomer abundances and the multinomial distribution to estimate two key parameters related to the biosynthesis. These parameters are 1) the dilution of the precursor immediately before biosynthesis and 2) the dilution of the newly synthesized product in the sampled compartment. Differentiated 3T3-L1 cells incorporated 2 mM [1,2-13C]acetate into triglyceride palmitate, yielding a spectrum of mass isotopomers of palmitate. The set of equations for the first nine isotopomers were solved for the two parameters using nonlinear regression. We found that precursor dilutions for acetate and glucose were constant over time, whereas the product dilution parameter increased with time, as expected for cells accumulating triglyceride palmitate. Mathematical procedures are presented for calculating 1) the predicted isotopomer fractional abundance values and 2) the correction for atoms other than the tracer atom in the mass ion.

Model equations for condensation biosynthesis using stable isotopes and radioisotopes.

Important syntheses in living systems occur by condensation reactions of the type nA----1B (where n is the number of A molecules needed to synthesize 1 molecule of B). Quantitative relationships for estimating the rate of synthesis of B from radioactive and stable isotope tracers are compared. With radioisotope tracers, only a single quantity is detected, the amount of radioactivity in B. In contrast, isotopes of varying mass produce multiple mass isotopomers B that are detected using mass spectrometry. The analysis demonstrates that the rate of synthesis of B is identifiable from stable isotope data but not from radioisotope data. This results because the isotopomer distribution of B at any time after tracer addition is a function of only the multinomial distribution representing the synthesis of B from n molecules of A and two parameters representing the fractional fluxes of isotopically enriched molecules to the sampled compartment of B. The model considers the possibility that the sampled compartment of B may not reach isotopic steady state during the experiment. A graphical method for obtaining initial estimates of the two parameters is presented.

Seroma prevention using fibrin glue in a rat mastectomy model.

Seromas are a frequent complication of mastectomy (17% to 53%) in humans and are correlated to skin flap elevation, lymphovascular interruption, and drainage into surgically created potential spaces. The use of intraoperative topical fibrin glue to reduce morbidity in rats undergoing radical mastectomies has been evaluated. A model consistently producing seromas was developed by radical mastectomy and lymphadenectomy in the Sprague-Dawley rat. A fibrin glue application procedure was tested using this model. The double-blinded protocol called for spray application of saline or fibrin glue to mastectomy wounds followed by sequential inspection and necropsy on postoperative days 5, 8, 11, and 14. Topical fibrin glue was shown to be statistically significant in decreasing the presentation of seromas following a radical mastectomy in the Sprague-Dawley rat.

A new, rapid safe method for local radiation of intrathoracic sites.

A technique for intraoperative, intrathoracic placement of afterloading catheters for post-thoracotomy radiation therapy is described. This technique offers speed and simplicity and requires no advance planning of radiation therapy. It uses materials that are readily available in operative theaters and radiation oncology units. No radiation exposure of operating room personnel is involved. It also avoids the mechanical problems of crimping and dislodgment associated with the traditional method of individual placement of small afterloading catheters for local radiation therapy. Isodose curves derived from radiographs showing postsurgical source positions demonstrated that it was possible to achieve a uniform therapeutic radiation dose distribution to the chest wall at the site of desired radiotherapy.

The influence of the surgical wound on local tumor recurrence.

Failure of a primary surgical treatment for cancer is often caused by recurrence of the tumor at the surgical site. The KHT mouse tumor system recapitulates this experience and provides a useful model to test strategies for reducing the incidence of local recurrence after surgical excision. There was an 82% local recurrence of the KHT tumor after surgery. A cell dilution assay indicated that it would require only 39 tumor cells injected into the wound site to result in the same (82%) incidence of tumors. This figure is in contrast to 340 cells required when the cells were injected into an unwounded flank. With the B16 melanoma in C57B1 mice and the Meth A sarcoma in BALB/c mice, the number of cells necessary to induce a tumor (TD/50) was also significantly reduced when the cells were injected into a surgical wound rather than into nonwounded tissue. The difference in cell number was interpreted as the result of the presence of growth factors derived from the traumatized tissue and the inflammatory cells at the wound site. Neither a 5 nor a 15 Gy dose of x-radiation delivered to the wound site immediately after surgical excision of the KHT tumor resulted in a significant reduction in the incidence of local recurrences. When the same doses of x-radiation were given immediately after injecting 36 KHT cells into a wound, no tumors developed. This difference was believed to have resulted from the hypoxic condition in the wound site and the presence of residual clonogenic tumor cells in a nonproliferating (radioresistant) state.

Seroma prevention using fibrin glue during modified radical neck dissection in a rat model.

Seroma is a frequent sequelae of neck dissection involving cervical lymphadenectomy. The incidence is correlated with flap elevation, lymphovascular interruption, and tissue removal. Current methods of resolving seroma, such as vacuum drainage, are not risk free. A novel approach to this problem was the use of intraoperative topical fibrin glue. A model producing seromas was developed by modified radical neck dissection on Sprague-Dawley rats. Forty rats underwent this procedure. Twenty rats were treated with saline solution (control group) and 20 were treated with fibrin glue. At necropsy on day 5, a significant reduction in the frequency of seroma was noted in the fibrin glue group. Seventeen of 20 control rats had seroma whereas only 2 of 20 experimental animals had serous collection. The Fisher exact statistical correlation revealed p less than 0.000002; therefore, the use of fibrin glue in this role merits further evaluation.

Mechanical performance of a work hardened pointed surgical wire.

The design and material properties of a surgical wire have been enhanced to improve its performance in surgery. One blunt end of the wire has been honed to a point to decrease the axial compressive force required to penetrate tissue. In addition, its sharp end has been work hardened to increase its yield strength. The mechanical performance of this wire has been characterized by a series of in vitro experiments and a clinical trial.

Heat welding for surgical sutures.

The purpose of this experimental study was to test the potential value of heat welding to cut the ends of knotted surgical sutures. This heat process resulted in knot security in a coated polyglactin suture with fewer throws than those needed for the same suture with knotted ends cut by scissors. Results of mechanical performance testing of the knotted sutures cut either by scissors or welding did not differ significantly. With refinements in the technical design of the heat source, suture welding may have important applications in surgery.

Bacteriologic evaluation of electric clippers for surgical hair removal.

Clipper blade assemblies for electric clippers used repeatedly without sterilization demonstrated high levels of bacterial contaminations that are potential sources of infection. A technique of sterilization of the clipper blade has been reported that eliminates the exogenous bacterial contamination.