Pseudomyxoma peritonei

Mucinous secretions are produced in diseases such as inflammatory bowel disease (e.g. ulcerative colitis and Crohn's disease),' cystic fibrosis,' ovarian cysts3 and different types of cancer.4,5 Many clinical benefits would be realised, including aiding both the dispersal of chemotherapy and removal of the mucin, if the viscosity of such secretions could be reduced.

Mucin is a viscous secretion produced by, and covering, many epithelial surfaces of the body, including those in the stomach, lung, intestine and cervix, where its effect is both protective and lubricant.6 Specialised epithelial cells secrete this mucin under strict genetic control.

The main advantages of performing the proposed assay in vitro rather than in vivo are cost-effectiveness, lack of patient trauma, and the ability to target the assay to the patient. For example, a small sample of mucin could be removed from the patient and tested against a panel of agents thought to be effective in reducing viscosity. Those that proved most effective in vitro then could be modified for use in patient treatment.

A good example of this is in cystic fibrosis (CF), where various mucolytic agents, including the enzymes DNase and hyaluronidase, have been used in an attempt to liquefy the mucinous secretion. DNase has been used in vitro to reduce the viscosity of sputum collected from CF patients, from a nonflowing viscous gel to a flowing liquid.' More recently, DNase (as Dornase cc; I mg/mL) has successfully improved respiratory function in CF patients, when given in a nebuliser.8 In contrast, hyaluronidase has been tested on mucin from patients with the mucin-secreting tumour pseudomyxoma peritonei (PMP), and found not to change the nature of the gelatinous material.'

N-acetyl-L-cysteine (20%) is another mucolytic agent that has been used to reduce mucin viscosity in sputum samples; proving to be extremely effective, with almost no associated complications. It is reported to achieve marked liquefaction within one minute, with a maximum effect seen after five to 10 minutes." More recently, nebulisation with 20% N-acetyl-L-cysteine was shown to reduce sputum viscosity in patients undergoing thoracotomy."

Viscosity is a measure of the resistive flow of a fluid, under the influence of gravity. When two fluids of equal volume are allowed to flow through identical capillary viscometers, a viscous fluid will take longer than a less viscous one. Currently, three main types of viscometer are available: a capillary viscometer, which measures the flow rate of a fluid through a narrow tube at a fixed temperature;" a plate viscometer, which uses torque on a rotating shaft to measure the resistance to flow;1,2 and a number of different instruments, operating in various ways, which, for example, measure the time taken for a steel ball to fall through a liquid.6

However, commercially available machines are both very expensive and require large volumes of sample, which is not always possible with biological material. Simpler methods, such as capillary flow or the falling ball-bearing method, make the production of replicates more difficult and time consuming because of the necessity to clean and sterilise the tube or ball-bearing between each replicate. Problems also could be encountered with less viscous solutions, as the time periods to be measured would be so small.

For these reasons, a new method of assessing the viscosity of a biological solution was devised. In this, the flow rate of a measured drop was timed as it travelled down an angled glass plate (Figure 1). The method proved easy to replicate as up to 10 replicates could be timed on one plate before the necessary sterilising and cleaning procedure. Less-viscous solutions could also be measured easily as the distance for the drop to fall could be increased so that timing was more manageable. The small volumes of mucinous fluid needed for this method will make it useful for many biological applications. Here, we test the method using mucinous ovarian cyst fluids.

Initial experimental work was performed on mucinous ovarian cyst fluids collected at laparotomy for ovarian cancer. Patients gave written consent for the samples to be removed during surgery, and Southern Derbyshire Ethics Committee gave ethical approval. Each cyst fluid sample was given a number in ascending order and the samples were stored at -20degC.

Dextrose (5% and 10%; Sigma Chemical Company, Poole, Dorset, UK), N-acetyl-L-cysteine (20%; Sigma), DNase (as Dornase (x [1 mg/mL]; Genentech, South San Francisco, CA, USA) and urea (6 molL; BDH, Poole, Dorset, UK) were tested in this system against a water control. Each agent was assessed at 37degC and 45degC. The higher temperature has been used in the chemotherapeutic treatment of patients with PMP."3 A physical agent (sonication) also was assessed for various time periods up to two minutes.

The mucinous fluid samples were thawed, a 540 gL sample of each was measured into an Eppendorf tube (Sarstedt, Leicester, UK) and 60 liL of either the test agent (10% v/v) or water was added. The resulting solution was vortex-mixed and then heated for 10 minutes at either 37degC or 45degC in a dry block heater (Unitek, supplied by Scientific Laboratory Supplies Ltd., Nottingham, UK). Viscosity was measured by timing the flow of 50 ltL samples of these solutions moving 10 cm down an angled (600) glass plate (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK), using a stopwatch (Casio Computer Company Ltd., Tokyo, Japan). This was repeated 10 times for each solution, after which the glass plate was washed, sterilised thoroughly, and given a final clean with 96% ethanol (BDH), to ensure a clean, non-sticky surface.

Statistical analysis was performed by pairedsample Student's t-test against the controls. A Bonferroni correction was applied to the results because of the number of Student's t-tests that had been performed.

All mucinous cyst fluids used in this study were taken from patients with either mucinous or seromucinous cystadenoma; and, even within this defined group, the consistency of fluid varied from watery to very mucinous. This was reflected by large differences in timings between the cyst fluids, as seen in Table 1 (sonication-untreated control).

The five agents tested produced variable results, showing either increased or decreased viscosity, probably due to the complex biological nature of the cyst fluids. Increasing the incubation temperature from 37degC to 45degC also showed variable effect. For each agent, little variation was seen between the ten replicates, as shown by the mean and standard deviation (SD) in Table 1.

After comparison using a paired-sample Students t-test, a Bonferroni correction was applied and the resultant highly significant decreases in relative viscosity are shown in Figure 2.

Of the agents tested, only 20% N-acetyl-L-cysteine decreased the viscosity of any of the cyst fluids significantly. Sonication decreased the viscosity of all of the cyst fluids at all of the times measured; however, after statistical analysis, only two of the cyst fluids showed decreased viscosity with sonication.

This novel method of assessing the viscosity of biological solutions was very simple to use, giving good reproducible replicates (Table 1). As might be expected of biological material, the samples tested proved very variable, yet the method coped well and it was possible to assess all the samples. The only chemical agent found to reduce the viscosity of some mucinous ovarian cyst fluids was 20% N-acetyl-L-cysteine; however, it would be easy to assess other chemical or physical agents using this technique.

Therefore, we suggest that this technique would be ideal to assess new chemical compounds thought to affect the viscosity of mucinous biological secretions. In order to aid the standardisation of the procedure, a primary standard of powdered bovine mucin (Sigma), prepared in aqueous solution at known concentration, could be used each time a new sample was analysed.

This project formed part of a New Researchers course attended by Mrs Linda Hubbold as part of the Trent Institutes New Researchers Training Programme. Mrs Hubbold would also like to thank Mr 1. V. Scott for collection of the samples and Southern Derbyshire Acute Hospitals NHS, Trust for funding this project.

References

1 Buisine MP, Desreumaux P, Debailleul V et al. Abnormalities in mucin gene expression in Crohn's disease. Inflamm Bowel Dis 1999; 5: 24-32.

2 Reas HW. The effect of N-acetylcysteine on the viscosity of tracheobronchial secretions in cystic fibrosis of the pancreas. J Pediatr 1963; 62: 31-5.

3 Giuntoli RL, Rodriguez GC, Whitaker RS, Dodge R, Voynow

JA. Mucin gene expression in ovarian cancers. Cancer Res 1998;58:5546-50.

4 Nguyen PL, Niehans GA, Cherwitz DL, Kim YS, Ho SB. Membrane-bound (MUCI) and secretory (MUC2, MUC3 and MUC4) mucin gene expression in human lung cancer. Tumour Biol 1996; 17: 176-92.

5 Finnie IA, Dwarakanath AD, Taylor BA, Rhodes JM. Colonic mucin synthesis is increased by sodium butyrate. Gut 1995; 36: 93-9.

6 Bansil R, Stanley E, LaMont JT. Mucin biophysics. Annu Rev Physiol 1995; 57: 635-57.

7 Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci USA 1990; 87: 9188-92.

8 Payne JN, Dixon S, Cooper NJ, McCabe CJ. The use of DNase in cystic fibrosis. Trent Institute for Health Services Research Guidance Notes 1996; 96(01): 1-16.

9 Carter J, Carson LF, Moradi MM, Adcock LA, Twiggs LB. Pseudomyxoma peritonei: a review. Int J Gynecol Cancer 1991; 1: 243-7.

10 Webb WR. Clinical evaluation of a new mucolytic agent, acetyl cysteine. J Thor Cardiovasc Surg 1962; 44: 330-43.

11 Gallon AM. Evaluation of nebulised acetylcysteine and normal saline in the treatment of sputum retention following thoracotomy. Thorax 1996; 51: 429-32.

12 Harding SE. The intrinsic viscosity of biological macromolecules. Progress in measurement, interpretation and application to structure in dilute solution. Prog Biophys Mol Biol 1997; 68: 207-62.

13 Sugarbaker PH, Ronnett BM, Archer A et al. Pseudomyxoma peritonei syndrome. Adv Surgery 1997; 30: 233-80.

L. M. HUBBOLD, C. M. GARNER and P. R. CHAKRABORTI

Oncology Research Laboratory, Derby Cancer Centre, Derby City General Hospital, Uttoxeter Road, Derby DE22 ME, UK

Copyright Royal Society of Medicine Press Ltd. 2001
Provided by ProQuest Information and Learning Company. All rights Reserved