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TEACHING IN THE LABORATORY

Vertebrate osmoregulation: a student laboratory exercise using teleost fish

¹ Department of Biology and Environmental Sciences, Western Connecticut State University, Danbury, Connecticut
² Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana

Address for reprint requests and other correspondence: P. Boily, Dept. of Biology and Environmental Sciences, Western Connecticut State Univ., 181 White St., Danbury, CT 06810 (E-mail: boilyp{at}wcsu.edu)

	Abstract

TOP Abstract Introduction MATERIALS AND METHODS TYPICAL RESULTS AND... APPENDIX REFERENCES

 
Here, we describe a laboratory experiment as part of an upper-level vertebrate physiology course for biology majors to investigate the physiological response of vertebrates to osmoregulatory challenges. The experiment involves measuring plasma osmolality and Na⁺-K⁺-ATPase activity in gill tissue of teleost fish acclimated to water of differing salinity. We describe results obtained using the widely available goldfish (Carassius auratus) and a common baitfish, the Gulf killifish (Fundulus grandis). The procedures described are generally applicable to other fish species, and they provide an alternative to the experimental use of humans or other mammalian species to investigate osmoregulation mechanisms. In addition to reenforcing the conceptual material covered in lecture, this laboratory exercise trains students in a wide range of laboratory and analytical skills, such as calculating and performing dilutions, pipetting, tissue sampling and homogenizing, preparing standard curves, conducting enzymatic assays, and analyzing and interpreting results. Typical student results are presented and discussed, as are common experimental and conceptual mistakes made by students.

Key words: chloride cells; mitochondrion-rich cells; ionoregulation; salinity; gills

	Introduction

TOP Abstract Introduction MATERIALS AND METHODS TYPICAL RESULTS AND... APPENDIX REFERENCES

 
COURSES IN ANIMAL PHYSIOLOGY typically include a discussion of the mechanisms involved in osmoregulation. Most experiments documented in laboratory textbooks and in journals such as this one are limited to the investigation of renal physiology using human or rodents as experimental subjects (10, 12), with two notable exceptions that describe laboratory experiments studying osmoregulatory mechanisms of amphibians (2, 11). While the use of humans or mammals is preferred and justified for some pedagogical contexts, safety, ethical, and legal concerns favor the use of the least sentient species that can provide satisfactory results. In fact, the objectives of many physiology courses can be satisfied, or enhanced, by laboratory experiments investigating the osmoregulatory capacity and mechanisms of nonmammalian vertebrates. Here, we describe an upper-level vertebrate physiology laboratory exercise using common teleost fish that supports and complements lecture material on general patterns and mechanisms of osmoregulation.

Many teleosts are excellent osmoregulators, being able to maintain a stable internal osmotic environment across a wide range of environmental salinity. Euryhalinity is especially common in diadromous species that migrate between saltwater and freshwater and brackish species that live in estuaries where salinity changes on tidal and seasonal cycles. One of the main sites of osmoregulation in teleosts are the mitochondrion-rich cells (MRCs), often referred to as "chloride cells," of the gill epithelia. Ion transport by these cells depends on the activity of Na⁺-K⁺-ATPase. This enzyme's activity is known to change in the gills of fishes when acclimated to water of different salinities. This change may be caused by an increase the number of MRCs, an increase in the enzyme activity of each cell, or a combination of both (5). The specific goal of the experiment is for students to address the following questions:

1. Are the fish used osmoregulators or osmoconformers? This question is addressed by measurements of blood plasma osmolality in fish acclimated to different salinities.
   
2. Does the osmoregulatory activity of the gill change when fish are acclimated to different salinities? This question is addressed by measurements of Na⁺-K⁺-ATPase activity of gill extracts.
   

	MATERIALS AND METHODS

TOP Abstract Introduction MATERIALS AND METHODS TYPICAL RESULTS AND... APPENDIX REFERENCES

 
Students work in groups of two or three. Students analyze data collected by all groups from all laboratory sections and write reports based on these results.

Materials
Equipment.
The following pieces of equipment are necessary:

• An osmometer capable of measuring the osmolality of 10-µl samples (1 for the class)
  
• Spectrophotometers (1 per group)
  
• Vortex mixers (1 per group)
  
• A microcapillary (hematocrit) centrifuge (1 for the class)
  
• A microcentrifuge (1 for the class)
  
• Analytical balances (mg accuracy) (1–3 for the class)
  
• Top-loading balances (0.01 g accuracy) (1–3 for the class)
  
• Tissue grinders (2-ml capacity) (1 per group)
  
• Adjustable pipettors (one 200-µl pipettor and one 1,000-µl pipettor per group)
  
• Stopwatches (1 per group)
  
• Aquariums with aeration and filtration equipment (at least 2 for the class; 1 for each salinity level)
  
• Computers with statistical and graphing software (at least 3 for the class)
  

Disposable supplies.
Disposable supplies include the following:

• Microcentrifuge tubes
  
• Microcapillary tubes
  
• Pipet tips
  
• Razor blades
  
• Parafilm
  
• Spectrophotometer cuvettes
  
• Solutions (see the APPENDIX)
  
• Ice buckets
  

Animals.
Although the goldfish (Carassius auratus) is generally considered to be stenohaline and restricted to fresh water environments, recent evidence demonstrate that it is in fact capable of tolerating brackish environments (13). This, combined with its widespread availability, makes it an excellent study model. Brackish baitfish species, such as the Gulf killifish (Fundulus grandis), can be easily obtained in bait shops in many coastal states and have the advantage of tolerating wider ranges of salinities (6); however, they are not as widely available as goldfish. Potentially a variety of other species could be used, as long as they are reasonably tolerant of salinity changes, available, and easy to maintain in the laboratory, and of appropriate size. With regard to size, we have found it difficult to sample tissues from fish smaller than 3–5 g. On the other hand, fish larger than 10–20 g require greater cost and space to maintain an appropriate number of individuals (30 fish).

In our experiments, goldfish or Gulf killifish were kept in 38-liter aquariums at a density of 10–20 fish/aquarium (depending on size) and fed commercial fish food. Water was aerated and filtered. The species used depended on their availability. Goldfish were purchased from local pet vendors, and they were supplied in freshwater. In the laboratory, they were randomly divided into two or more groups and acclimated to water having osmolalities from near freshwater (10 mosM/kg) up to 350 mosM/kg. Gulf killifish were purchased from a local bait dealer, and they were provided in water from their habitat (300–600 mosM/kg). In the laboratory, they were randomly divided into two or more groups and acclimated to waters having osmolalities from 100 to 1,000 mosM/kg (seawater osmolality is 1,200 mosM/kg). Ideally, one of the chosen salinities would be approximately isosmotic to fish plasma (300–400 mosM/kg). For both species, water osmolality was changed at a rate of 50–200 mosM/kg every 3–4 days by performing partial water changes with either dechlorinated tap water or artificial seawater made with Instant Ocean (Aquarium Systems, Mentor, OH). Fish were sampled at least 1 wk after reaching the desired acclimation salinity. We have not experienced any fish mortality using this acclimation protocol.

Typically, we have six groups of 2–3 students/laboratory section, with 2 laboratory sections/semester. Each group of students samples 1 fish from 2 different salinity levels, leading to a sample size of 24 fish/semester. The exercise can be completed in one 3-h laboratory session; suggestions for extending the exercise to two 3-h sessions are given in Additional Experiments, some of which increase the number of fish used.

Experimental Procedures
The activity of Na⁺-K⁺ ATPase in gill extracts is determined using an enzymatically coupled assay previously described (8). The ADP produced by gill extracts is used by pyruvate kinase along with phosphoenol pyruvate to form pyruvate and ATP, and the pyruvate is reduced by lactate dehydrogenase to lactate with the concomitant oxidation of NADH to NAD⁺. Therefore, the rate of ADP formation equals the rate of NADH disappearance, which is monitored as a decrease in absorbance at 340 nm. The first step in this procedure is to generate a standard curve relating the amount of ADP to absorbance at 340 nm.

Standard curve.
Students prepare five microcentrifuge tubes containing 250 µl of 0, 0.25, 0.5, 0.75, or 1.0 mM ADP, using a 1 mM ADP stock solution and homogenization buffer as the diluent (all solutions are described in the APPENDIX). Students calculate their own dilutions, which are verified by the instructor before proceeding.

Students prepare an additional set of 12 microcentrifuge tubes (6 duplicates) containing 100 µl of each of dilution made above and 1 ml of ADP assay solution. The tubes are mixed by vortexing, incubated at room temperature for 5 min, and measured for absorbance at 340 nm. The relationship between absorbance (average of duplicates) and the amount of ADP in each tube (0, 20, 50, 75, and 100 nmol) is plotted to verify linearity, and the slope (in absorbance units/nmol ADP) is calculated (Fig. 1). This slope is later used to calculate Na⁺-K⁺-ATPase activity of the samples. Students must complete the standard curve part of the laboratory exercise before proceeding to the next step.

View larger version (8K): [in this window] [in a new window]   Fig. 1. Typical standard curve of absorbance as a function of the amount of ADP contained in each tube. The slope of the regression is –0.0044 absorbance units/nmol (R2 = 0.98).

Immediately after the blood sample is obtained, the fish is decapitated, and the gills are quickly dissected out of the branchial chamber. The gills are placed on an ice-cold dish, and the gill filaments (pink epithelium) are dissected from the gill arches (translucent cartilage) with microdissecting scissors. The gill filaments are weighed (to the nearest mg) and placed in a tissue grinder containing 1.0 ml of ice-cold homogenization buffer. The experiment works well with 0.05 g of tissue. The tissue is homogenized by hand, on ice, for 2 min. The homogenate is transferred to a microcentrifuge tube and centrifuged for 1 min at 6,000 rpm. The supernatant is transferred to a new microcentrifuge tube and kept on ice. The fish carcass is weighed at a convenient time. The blood sampling, gill dissection, and extract preparation are repeated on a fish from a different salinity. Depending on student abilities, the two samples can be prepared at the same time or sequentially. Care must be taken to keep the extracts on ice.

Na⁺-K⁺-ATPase assay on gill homogenates.
The rate of Na⁺-K⁺-ATPase activity in the homogenates is determined by the difference in the rate of ADP formation in the presence or absence of ouabain, a specific Na⁺-K⁺-ATPase blocker.

The Na⁺-K⁺-ATPase assay is carried out as follows: 100 µl of gill homogenate is pipetted into a spectrophotometer cuvette. Working quickly, 1.0 ml of ADP assay solution (same solution used for the standard curve) is added, and the cuvette is covered with Parafilm, mixed by inverting it several times, and placed in the spectrophotometer (340 nm, blanked with water). The absorbance is measured immediately and every 10 s for the next 60 s. This procedure is repeated with the same gill extract but using an ADP assay solution that contains ouabain (0.5 mM). Each measurement is done in duplicate, for a total of 4 assays/gill homogenate (2 assays using a solution without ouabain and 2 assays using a solution with ouabain).

For each assay, the slope of the relationship of absorbance as a function of time (absolute value in absorbance units/min) is calculated. For each sample, the average slope obtained for the duplicate assays with ouabain (Slope_AO) is subtracted from the average slope obtained for the duplicate assays without ouabain (Slope_A). This difference in the slopes (Fig. 2) is divided by the slope of the standard curve (Slope_STD) previously calculated (absolute value in absorbance units/nmol ADP) to obtain the rate of Na⁺-K⁺-ATPase activity (nmol ADP/min), as follows:

(1)

This rate of enzymatic activity must be standardized for the mass of gill filament tissue in each assay and converted to international units of enzymatic activity (units or µmol/min). First, the mass of gill epithelia used in each assay (M_Ga) is calculated, taking into account the mass (M_Gh) and volume (V_Gh; calculated assuming a tissue density of 1g/ml) of the gill epithelia homogenized, the volume of the homogenization buffer (V_HB; 1 ml), and the volume of supernatant used in each assay (V_S; 0.1 ml), as follows:

(2)

The rate of enzymatic activity (in U/g) is then calculated as follows:

(3)

View larger version (10K): [in this window] [in a new window]   Fig. 2. Typical changes in absorbance over time of ADP assays using solutions without ouabain [solution A (Sol. A): slope = –0.070 absorbance units/min, R2 = 0.98] or with ouabain [solution B (Sol. B): slope = –0.043 absorbance units/min, R2= 0.99].

(4)

(5)

(6)

Data analysis.
By combining data from all laboratory sections, the class typically has a sample size of 24 fish, with 12 fish from each salinity level. The differences in plasma osmolality and in Na⁺-K⁺-ATPase activity between treatments are evaluated using an unpaired (independent) t-test. If more than two treatments are used (as in the case below, where we have 3–4 different salinities), multiple comparison tests must be used; we use the "false discovery rate procedure" (4). If necessary, Na⁺-K⁺-ATPase activity rates are log transformed to satisfy assumptions of parametric statistics.

Common Technical Mistakes Made by Students
This laboratory experiment involves many steps, using many different solutions, leading to a high probably of mistakes. The numbers of calculations and units used throughout the experiment can also overwhelm students. We provide students with a step-by-step, simplified protocol that includes worksheets for calculations. In addition, all solutions should be clearly labeled with color-coded tape.

The most common technical mistakes that we observed are related to basic laboratory skills, especially the proper use of pipettors and the calculation of dilutions. We review basic laboratory procedures during the prelaboratory introduction and verify the students’ dilution calculations before they proceed to prepare their ADP standard curve.

The dissection of gills must be done quickly and carefully. It is important to avoid the skeletal elements of the gill arch because this would contribute to the tissue mass but not to Na⁺-K⁺-ATPase activity. Students are instructed that very little tissue is needed (0.05 g) and that is better to have slightly less tissue dissected quickly and carefully than to have a larger mass of poorly dissected tissue.

Because of the potential hazard of breaking capillary tubes and the sensitivity of the osmometer to user error, an instructor handles blood samples after collection (including centrifugation, obtaining plasma, and measuring osmolality), with the students taking turn observing the procedure.

In the Na⁺-K⁺-ATPase assay, NADH is in limited supply. Therefore, the rate of reaction will slow down as NADH is consumed, resulting in poor linearity of the assay if students are too slow to start the absorbance readings. We ask the student to graph the results to verify linearity when they calculate the slope of the relationship of absorbance as a function of time. On occasion, high enzyme activities will yield slopes that are not linear over the entire 60 s of the assay. On these occasions, students are instructed to eliminate the final data points to achieve a linear enzyme rate.

	TYPICAL RESULTS AND INTERPRETATIONS

TOP Abstract Introduction MATERIALS AND METHODS TYPICAL RESULTS AND... APPENDIX REFERENCES

 
Goldfish
Figure 3 shows the combined results obtained by students using goldfish exposed to 0 and 240 mosM/kg water during one semester and to 60 and 350 mosM/kg water for the other semester. Plasma osmolality was significantly lowest in goldfish acclimated to 0 mosM/kg water, highest in goldfish acclimated to 351 mosM/kg water, and intermediate for fish acclimated to 60 and 240 mosM/kg water. These results suggest that goldfish are imperfect osmoregulators, being able to maintain stable plasma osmolality of 300–305 mosM/kg in 60 and 240 mosM/kg water, with some variation in plasma osmolality at lower and higher salinities. Gill Na⁺-K⁺-ATPase activities were lowest in goldfish acclimated to 240 mosM/kg water. Higher activities were measured in goldfish acclimated to lower (0–60 mosM/kg) and higher (351 mosM/kg) salinities. At low salinities, the greater gill Na⁺-K⁺-ATPase activities presumably help compensate for ion loss to a dilute medium, although this compensation is incomplete (as indicated by decreased plasma osmolality in the 0 mosM/kg water). At the higher salinity, the modest increase in Na⁺-K⁺-ATPase activity might be an attempt to restore plasma osmolality to values measured at intermediate salinities (300–305 mosM/kg) or to eliminate ions coming from other sources (e.g., diet). Taken together, the results of plasma osmolality and gill Na⁺-K⁺-ATPase activities are consistent with recent observations showing that goldfish appear to be more euryhaline than previously thought, being able to survive exposure to water ranging from freshwater to approximately one-third the osmolality of seawater (13).

View larger version (8K): [in this window] [in a new window]   Fig. 3. Plasma osmolality (top) and Na+-K+-ATPase activity (bottom) of gill tissue in goldfish [Carassius auratus (C.a.)] acclimated to different water osmolalities. Values are presented as means ± SD. A,B,CValues with different letters differed significantly according to a "false discovery rate procedure" statistical test.

View larger version (8K): [in this window] [in a new window]   Fig. 4. Plasma osmolality (top) and Na+-K+-ATPase activity (bottom) of gill tissue in Gulf killifish [Fundulus grandis (F.g.)] acclimated to different water osmolalities. Values are presented as means ± SD. A,BValues with different letters differed significantly according to a "false discovery rate procedure" statistical test.

Second, students usually presume that Na⁺-K⁺-ATPase activities should increase with increasing water salinity, which is not the case for goldfish used in this study. Instead, the Na⁺-K⁺-ATPase activities reflect the osmotic gradient, i.e., the difference between internal (plasma) and external (water) osmolality, which can be demonstrated by performing a regression analysis of mean Na⁺-K⁺-ATPase activity rates as a function of the mean absolute difference between plasma and water osmolality for both species (Fig. 5). Deviations from this line are relatively small and may be due to other routes of ion gain or loss by these fish. For example, the kidneys and digestive tract are also involved in osmoregulation of teleosts, especially for the excretion of divalent ions.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Regression (R2 = 0.93) of mean Na+-K+-ATPase activities of gill epithelia as a function of the mean absolute difference between plasma and water osmolality for goldfish (C.a.) and Gulf killifish (F.g.).

Protein concentration of gill homogenates.
Another option for extending this laboratory exercise is to measure the protein concentration of the remaining gill homogenates (0.6 ml of solution should remain and may be frozen for later protein analysis). Protein measurements allow students to express the rates of Na⁺-K⁺-ATPase as specific activity (in U/mg protein) rather than based on wet tissue mass. Measurements of protein concentration can easily be done using previously published methods (3), and students gain more experience with diluting, pipetting, constructing standard curves, and analyzing data. In our experience, however, we have found that expressing the rates of Na⁺-K⁺-ATPase activity per milligram of protein did not result in lower variance than if expressed per gram of tissue. Therefore, we no longer have students perform this additional step.

Whole animal metabolic rate.
A potential followup or complementary question to this experiment is to ask if the changes in the ion pumping activity result in differences in the overall energy expenditure of the individual. Simple methods to measure metabolic rates of fish are well documented (1), and measurements could be done the week before the fish are killed for the experiments described above. Recent studies (7, 9) have suggested that the metabolic cost of ion regulation account for <10% of the standard metabolic rate in Fundulus heteroclitus, a closely related killifish from estuaries of Atlantic coast, and <5% in cutthroat trout (Oncorhynchus clarki clarki). Given the sensitivity of metabolic rate to disturbance, activity, feeding, photoperiod, and other factors that are difficult to control in a student laboratory, it is unlikely that such small differences would be measurable.

	APPENDIX

TOP Abstract Introduction MATERIALS AND METHODS TYPICAL RESULTS AND... APPENDIX REFERENCES

 
Solutions needed for two laboratory sections of six groups of students are as follows.

Imidazole Buffer
Imidazole buffer is used at a concentration of 50 mM and is used as the buffer for all solutions below. It is made by dissolving 1.36 g imidazole [formula weight (FW) = 68.08] in 350 ml water and titrated with HCl to pH 7.5, and the volume is adjusted to 400 ml with water.

Homogenization Buffer
Homogenization buffer is composed of 150 mM sucrose and 10 mM EDTA in 50 mM imidazole and is used for preparing ADP standards and for tissue homogenization. It is made by adding 4.11 g sucrose (FW = 342.3) and 0.298 g disodium EDTA (FW = 372.2) to 80 ml imidazole buffer. After the solution has been stirred until solids dissolve, 0.08 g sodium deoxycholate is added to obtain a 0.1% solution. It must be refrigerated and used within 4 days. Each group receives 4 ml of homogenization buffer.

ADP Standard Solution
ADP standard solution is used at a concentration of 1 mM and is used to make various standards. It is made by adding 0.0075 g ADP (FW = 501) to 15 ml homogenization buffer. It must be kept frozen until immediately before use. Each group receives 1 ml of ADP standard solution.

Salt Solution
Salt solution is composed of 189 mM NaCl, 42 mM KCl, and 10.5 mM MgCl₂ in 50 mM imidazole and is used for the assay solution below. It is made by adding 0.884 g NaCl (FW = 58.44) and 0.251g KCl (FW = 74.55) to 80 ml imidazole buffer. After the solution has been stirred until solids dissolve, 0.84 ml MgCl₂ solution (1 M) is added. It must be refrigerated and used within 4 days.

Assay Solutions
Assay solutions are made with or without ouabain and are used to measured Na⁺-K⁺-ATPase activity. They must be made the day of the labotory exercise. Assay solutions are made by adding the following to 240 ml imidazole buffer:

• 0.1276 g PEP (FW = 190) for a concentration of 2.8 mM
  
• 0.0926 g ATP (FW = 551.1) for a concentration of 0.7 mM
  
• 0.0374 g NADH (FW = 709) for a concentration of 0.22 mM
  
• 960 units lactate dehydrogenase for a concentration of 4 U/ml
  
• 1200 units pyruvate kinase for a concentration of 5 U/ml
  

To these solutions, add 80 ml salt solution. Each group receives 20 ml of a given assay solution. To the remaining 80 ml assay solution, 0.0292 g ouabain octahydrate (FW = 728.77) is added to obtain a final concentration of 0.5 mM. Each group receives 6.5 ml of assay solution with ouabain.

	Acknowledgments

 
We thank all the Vertebrate Physiology students of the University of New Orleans who have participated in this laboratory exercise.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication May 31, 2007. Accepted for publication August 24, 2007.

	REFERENCES

TOP Abstract Introduction MATERIALS AND METHODS TYPICAL RESULTS AND... APPENDIX REFERENCES

 

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3. Brown RE, Jarvis KL, Hyland KJ. Protein measurement using bicinchoninic acid: elimination of interfering substance. Anal Biochem 180: 136–139, 1989.[CrossRef][ISI][Medline]
4. Curran-Everett D. Multiple comparisons: philosophies and illustrations. Am J Physiol Reg Integr Comp Physiol 279: R1–R8, 2000.[Abstract/Free Full Text]
5. Evans DH, Piermarini PM, Choe KP. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85: 97–177, 2005.[Abstract/Free Full Text]
6. Griffith RW. Environment and salinity tolerance in the genus Fundulus. Copeia 1974: 319–331, 1974.[CrossRef]
7. Kidder GW III, Petersen CW, Preston RL. Energetics of osmoregulation: I. Oxygen consumption by Fundulus heteroclitus. J Exp Zool 305A: 309–217, 2006.
8. McCormick SD. Methods for nonlethal gill biopsy and measurement of Na⁺,K⁺-ATPase activity. Can J Fish Aquat Sci 50: 656–658, 1992.
9. Morgan JD, Iwama GK. Energy cost of NaCl transport in isolated gills of cutthroat trout. Am J Physiol Reg Integr Comp Physiol 277: R631–R639, 1999.[Abstract/Free Full Text]
10. Tharp GD, Woodman DA. Experiments in Physiology (9th ed.). San Francisco, CA: Cummings, 2007.
11. Thurman CL. The active transport of sodium in frog skin. In: Laboratory Manual for Physiology, edited by Silverthorn DU, Johnson BR, Mills AC. San Francisco, CA: Cummings, 2005, p. 785–800.
12. Walker RL, Olson ME. Renal function in the laboratory rat: a student exercise. Adv Physiol Educ 13: 49–55, 1995.
13. Schofield PJ, Brown ME, Fuller PL. Salinity tolerance of goldfish Carassius auratus L., a non-native fish in the United States. Florida Scientist 69: 258–268, 2006.

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Boily, P. Articles by Williamson, L. A. C. Search for Related Content PubMed PubMed Citation Articles by Boily, P. Articles by Williamson, L. A. C.