Dietary Conjugated Linoleic Acid Does Not Adversely Affect Bone Mass in Obese fa/fa or Lean Zucker Rats

 

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Laura L. Burr*,
Carla G. Taylor and
Hope A. Weiler*1

^* School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada; and Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada

¹ School of Dietetics and Human Nutrition, McGill University, 111 Lakeshore Road, Ste-Anne-de-Bellevue, QC H9X 3V9, Canada. E-mail: hope.weiler{at}mcgill.ca

	Abstract

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Conjugated linoleic acid (CLA) elevates body ash in healthy animals. The objective of the present study was to determine if single or mixed CLA isomers improve bone mass in an obese and hyperinsulinemic state. Male (n = 120) lean and obese fa/fa Zucker rats (age, 6 weeks) were randomized to 8 weeks on a control diet or to 0.4% (w/w) cis-9, trans-11 CLA (Group 1); 0.4% (w/w) trans-10, cis-12 CLA (Group 2); 0.4% (w/w) cis-9, trans-11 CLA and 0.4% (w/w) trans-10, cis-12 CLA (Group 3); 0.4% (w/w) cis-9, trans-11 CLA, 0.4% (w/w) trans-10, cis-12 CLA, and traces of other CLA isomers (Group 4); and 0.4% (w/w) cis-9, trans-11 CLA, 0.4% (w/w) trans-10, cis-12 CLA, and 0.3% (w/w) other CLA isomers (Group 5). Bone area (BA), bone mineral content (BMC), and bone mineral density (BMD) of the whole body, spine, and femur were measured at baseline (6 weeks) and at 14 weeks of age. Effects of genotype, diet, and genotype x diet interactions were assessed using factorial analysis of variance. At 6 and 14 weeks, whole-body BA and BMC were lower in lean rats compared with fa/fa rats. Similarly, at 14 weeks, fa/fa rats had a higher spine and femur BMD despite a lower femur weight. The fa/farats in Groups 4 and 5 had higher adjusted whole-body BMC comparedwith Group 3, but not with Group 1, Group 2, or the control.In lean rats, Group 3 had a greater adjusted whole-body BMCthan Groups 1 and 2, but not Group 4, Group 5, or the control.Thus, commercially available CLA mixtures and single CLA isomersdo not affect bone mass in a hyperinsulinemic, obese state.

Keywords: conjugated linoleic acid, bone mass, obesity, hyperinsulinemia, Zucker rat

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 

Polyunsaturated fatty acids (PUFAs), including conjugated linoleic acid (CLA), are recognized as nutrients involved in bone mineral homeostasis (1). CLA is a group of positional and geometric isomers of linoleic acid (2), of which the trans-10, cis-12 (t10, c12) and the cis-9, trans-11 (c9, t11) CLA isomers are sold commercially and available in mixed or pure forms (3). Research has heightened interest in CLA as a positive modulator of several health outcomes associated with excess body weight. Because CLA may be recommended therapeutically to overweight individuals, including young adults and adolescents (4, 5),there is a need to clarify whether CLA improves or reduces bonemass during a period of bone mineralization and consolidation.

Animal studies have provided evidence that CLA may play a role in bone mineralization and turnover. In general, the effect of CLA on bone mass appears to be dependent on the CLA isomers included in the diet (6), the dietary total n-6 to n-3 PUFA ratio (7), and the period of life when CLA is fed (8). The effect of CLA on bone also may be dose dependent (9). A CLA mixture containing many isomers was observed to have no effect on bone mass (8), but supplementing the diet primarily with t10, c12 CLA isomers elevated body ash in mice (6). Studies that observed positive effects of CLA on bone mass used a high n-6 to n-3 PUFA ratio for the dietary lipid composition (8–11), whereas a lower n-6 to n-3 PUFA ratio frequently was used during studies in which CLA had no effect (7, 12, 13). Studies conducted during periods of rapid growth have found a response in bone to CLA treatment. A positive effect of CLA on bone mass has been observed in male chicks fed butter fat from birth (10) and in growing male and female mice (6, 8, 11). Research conducted during later stages of growth in swine, however, suggests that CLA has no effect on bone mass (14, 15). A recent epidemiologic study conducted with postmenopausal women found that dietary CLA intake was positively associated with forearm bone mineral density (BMD; Ref. 16), but other studies in adults (17–19) have foundno effect of CLA on biochemical markers of bone resorption andformation or osteoporotic risk factors. Based on these studies,CLA might best be used to enhance bone mass during childhoodor adolescence, a time of continued bone growth and mineralization.

Osteoporosis may be a health risk (20) for the growing number of children and young adults in North America who are overweight or obese (21–23). In adults, BMD is positively associated with body weight (24, 25), but the protective effect of excess body fat on bone mass appears to diminish as body weight increases (26). In children, excess body weight (or, more specifically, fat mass) reduces bone mineral content (BMC; Refs. 27, 28) and increases the risk of bone fracture (29, 30). Because CLA has been reported in some studies to reduce body weight (31), dietary CLA may be useful in achieving a body weight that is supportive of optimal bone mineralization. Juvenile obesity also is associated with the development of hyperinsulinemia and type 2 diabetes mellitus in childhood (32, 33). Elevated bone mass is associated with hyperinsulinemia in adults (34, 35), but the effects onbone in the young are unknown.

Because it is premature to test for benefits of CLA on bone mass in children, the primary objective of the current study was to determine the effect of feeding c9, t11 or t10, c12 CLA isomers individually (0.4% w/w, where w/w is g/kg diet x 100), in combination (0.8% w/w CLA isomers), or in a mixture with other CLA isomers (0.8%–1.1% w/w CLA isomers) to young, male Zucker rats. The fa/fa Zucker genotype is an obese, leptin receptor–deficient model that exhibits hyperinsulinemia without hyperglycemia (36). This model also has been confirmed to be representative of juvenile obesity and bone metabolism (37). The fa/fa rat has reduced long bone longitudinal growth and whole-body bone mass, with lower bone formation and higher resorption, than lean Zucker rats by 12–24 weeks of age (37, 38). The fa/fa genotype, however, is responsive in bone to intervention, because previously, an exercise regime elevated bone mass between 3–6 months of age (39). Because of the potential of CLA to be a nutraceutical in the management of obesity and type 2 diabetes (40, 41), determining the effectof CLA isomers on bone mass in an obese, hyperinsulinemic modelis a necessary investigation to further ascertain the benefitsand side effects of CLA treatment.

	Materials and Methods

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Animals and Diet.

All animal care procedures were based on guidelines from the Canadian Council on Animal Care (42) and approved by the Universityof Manitoba Fort Garry Campus Protocol Management and ReviewCommittee.

Male lean and fa/fa Zucker rats were purchased from Harlan (Indianapolis, IN) at 5 weeks of age. Because of the potential confounding variables associated with female sex hormones and differences in pubertal onset between males and females, only male rats were used. Rats were housed individually in stainless steel, wire-bottomed cages in a controlled environment of 55% humidity and 21°–23°C with a 14:10-hr light:dark cycle. Rats followed longitudinally were acclimatized on a control diet for 5–9 days, and test diets were randomly assigned (n = 10 rats/diet group) and fed ad libitum for 8 weeks. Feed intake (corrected for spillage) and rat weight was measured weekly. To provide for baseline values, four lean and five fa/farats were terminated at 6 weeks of age (before receiving thetest diet). These rats underwent the same analysis as thosein the treatment groups (description follows).

All diets were based on the AIN-93G formulation and were nutritionally adequate for normal rat growth and development (43). Each diet contained 8.5% (w/w) lipid (Table 1). The control diet contained soybean oil exclusively. Test diets were designed to provide 0.4% (w/w) c9, t11 and/or t10, c12. Groups 1 and 2 received 0.4% (w/w) of pure c9, t11 and t10, c12, respectively, whereas Group 3 received both CLA isomers (0.4% w/w c9, t11 and 0.4% w/w t10, c12) or 0.8% (w/w) CLA isomers. Groups 4 and 5 also received both CLA isomers (0.4% w/w c9, t11 and 0.4% w/w t10, c12), but because of the presence of other CLA isomers in the oils used for these diets, those groups received 0.8% and 1.1% (w/w) CLA isomers, respectively. The two primary CLA isomers in the diets were chosen based on evidence that both are more effective at altering body composition than other CLA isomers and that both are present in commercially available CLA supplements. The amount of CLA added to each diet was determined based on a pilot study that indicated 0.4% (w/w) c9, t11 and t10, c12 improved lipid metabolism (44) and on previous research that found a positive effect of CLA on calcium metabolism using 0.45% (w/w) c9, t11 and 0.47% (w/w) t10, c12 (13).

At the end of the 8-week study duration, rats were euthanized by CO₂ asphyxiation and cervical dislocation for trunk bloodcollection.

Dual-Energy X-Ray Absorptiometry and Femur Measurements.

Analysis of bone mass was performed using dual-energy x-ray absorptiometry (DXA; small animal software; 4500A; Hologic, Inc., Bedford, MA), because DXA has been confirmed to be an appropriate method to evaluate body composition in the adult rat (45). DXA also was used with a high degree of accuracy and precision in mice weighing less than 100 g (46), and the small animal software was shown to be valid for use with young rodents (47), such as the baseline rats weighing 130–170 g. Scans were performed on frozen carcasses to measure whole-body bone area (BA), BMC, and BMD. Whole-body BMC was adjusted for the weight of the rat (g/kg) because of the large difference in body mass between the two genotypes. Regional scans were performed using high-resolution software to determine BA, BMC, and BMD for vertebrae 1–4 of the lumbar spine and the right femur in situ (48). Following DXA scanning, the right femur was removed, cleaned of soft tissue, weighed, and measured in triplicate to the nearest 0.01 mm for femur length, neck, proximal femur epiphysis width, diaphysis width, and knee width (49, 50). DXA also was used to measure the BA, BMC, and BMD of excised femurs by placing the bone in a water bath aligned in an anteroposterior position with 3 cm of water covering the femur (51).

Bone Mineral and Biochemistry.

Femur calcium and phosphorus concentrations were measured using inductively coupled plasma–optical-emission spectroscopy (Varian Liberty 200; Varian, Mississauga, ON, Canada) after digestion in nitric acid (52). Femurs were dried at 85°Cfor 72 hrs in glass test tubes. Concentrated, trace metal–gradenitric acid (1 ml) was added, and after complete digestion ofthe bone (72 hrs), deionized water was added to each tube toachieve a final volume of 20 ml (final concentration of 5% [v/v]nitric acid). No difference was observed for bone mass amongthe diet groups, thus serum osteocalcin was only measured inthe control diet groups for each genotype using an ELISA (Rat-MidOsteocalcin; Osteometer BioTech A/S, Herlev, Denmark).

Statistics.

A factorial analysis of variance was used to determine differences among groups (genotype x diet; P < 0.05). Body weight and food intake were tested for covariance with other variables but were ruled out after no further information was gained from this more complex model. When appropriate, least squares means testing was used to determine differences (P < 0.05) amongdiet groups. All results are expressed as the mean ±SEM.

	Results

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Baseline Measurements.

Six-week-old fa/fa rats had a higher body weight (169 ± 4 vs. 131 ± 3 g; P < 0.0001) and a shorter tail length (12.7 ± 0.2 vs. 14.0 ± 0.1 cm; P < 0.0001) compared with lean rats, but no differences in body length were found between genotypes (16.5 ± 0.2 vs. 16.9 ± 0.2 cm; P = 0.09). The fa/fa rats had a greater whole-body BA and BMC than lean rats, but BMD and regional scans of the lumbar spine (Table 2) did not differ significantly. Excised (Table 2) and in situ measurements of femur BA (0.701 ± 0.035 vs. 0.721 ± 0.037 cm²), BMC (0.117 ± 0.009 vs. 0.129 ± 0.008 g), and BMD (0.166 ± 0.011 vs. 0.180 ± 0.010 g/cm²) were not statistically different (P > 0.05) between fa/fa and lean rats. Femur dry weight did not differ between genotypes. Femur length, neck width, proximal femur epiphysis width, and knee width were lower in fa/fa rats than in lean rats, but diaphysis width as well as calcium and phosphorus concentrations were greater in the fa/fa rats.

Body Size, Tail Length, and Feed Intake.

Fourteen-week-old fa/fa rats had a higher body weight than lean rats (547 ± 5 vs. 327 ± 2 g; P < 0.0001). Although it failed to reach statistical significance, a 4% difference in body weight was found among lean diet groups (328 ± 5 for control vs. 317 ± 6 to 336 ± 7 g for CLA diet groups). For fa/fa diet groups, a 2%–5% difference was found in body weight (560 ± 13 for control vs. 533 ± 14 to 567 ± 18 g for CLA diet groups). The fa/fa rats had shorter tail lengths (16.6 ± 0.1 vs. 18.4 ± 0.1 cm; P < 0.0001) than lean rats. No differences were found in tail length among diet groups or in body length between genotypes (21.9 ± 0.1 vs. 21.8 ± 0.1 cm; P =0.2). The fa/fa rats had a greater feed intake over the 8-week study period than lean rats (1.52 ± 0.01 vs. 0.98 ± 0.01 kg; P < 0.0001). The fa/fa rats on the control diet and in Groups1, 3, and 4 ate significantly more than those in Group 2 (Fig.1). Within the lean treatment groups, Group 5 ate less thanother diet groups.

Bone Mass.

Whole-body BA and BMC were greater in the fa/fa rats than in lean rats (Table 2). After correcting for body weight (adjusted BMC), the lean rats displayed greater whole-body BMC (Fig. 2). Despite no significant difference in body weight between diet groups, lean rats from Group 3 had a greater adjusted BMC than those in Groups 1 and 2 but did not significantly differ from those in Group 4, Group 5, and the control. The fa/fa rats in Groups 4 and 5 had a higher adjusted BMC than fa/fa rats in Group 3 but did not significantly differ from those in Group 1, Group 2, and the control. BMD did not differ between genotypes or diet groups, but a trend toward higher whole-body BMD was observed in the lean rats (P = 0.06; Table 2).

No effects of diet were observed for DXA measures of the lumbar spine or femur; thus, only genotype differences are reported. Regional scans of the lumbar spine indicated greater BMD in the fa/fa rats than in the lean rats, but no differences were observed between fa/fa and lean rats for in situ DXA scans of the right femur (BA: 1.12 ± 0.05 vs. 1.00 ± 0.04 cm²; BMC: 0.378 ± 0.180 vs. 0.391 ± 0.150 g; BMD: 0.378 ± 0.010 vs. 0.350 ± 0.010 g/cm²) and lumbar spine BA and BMC (Table 2). Excised femurs from fa/fa rats exhibiteda lower BA and greater BMD, but not BMC, than lean rats (Table2).

Morphometric Measurements, Mineral Analysis, and Biochemistry.

No significant effects of diet were observed for morphometric measurements, mineral analysis, or biochemistry; thus, only genotype effects are reported. The fa/fa rat femurs had a lower dry weight than those from lean rats and shorter femurs with smaller measurements for the femoral neck, proximal femur epiphysis, and knee width (Table 2). Diaphysis width was greater in the fa/fa rats than in the lean rats.

No differences were observed between genotypes in femoral calcium concentration. Femoral phosphorus concentration was greater in fa/fa rats than in lean rats (Table 2). Serum osteocalcin concentration did not differ significantly between genotypes (66.7 ± 8.3 vs. 60.4 ± 6.8 nM; P = 0.50), indicatingno detectable difference in osteoblast activity or bone modeling.

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

 

The rising rate of obesity and associated metabolic abnormalities among the adolescent population has severe negative health consequences. CLA is suggested to reduce fat mass and to improve insulin sensitivity in overweight, young individuals (4), whereas the effects of CLA on bone mass in young humans is presently unknown. Childhood, adolescence, and young adulthood, however, are periods of life in which optimal bone mineralization is critical for lifelong bone health (53). Therefore, an investigation was warrantedto determine the effect of CLA on bone health in a young, obesestate. The present study demonstrates that CLA has no positiveor adverse effects on bone mass in a growing model of obesityand insulin resistance. This finding is an important contributionto CLA research, because it indicates that therapeutic CLA treatmentdoes not adversely affect bone mineral accretion during an adolescentstage of growth in a male rodent model.

At 14 weeks of age, the fa/fa rats in the present study displayed genotypic manifestations, including obesity and hyperinsulinemia, without hyperglycemia (data not shown). Despite greater body weight, the fa/fa rats demonstrated lower adjusted whole-body BMC and reduced femoral bone growth (indicated by morphometry) in comparison to lean rats, a finding that is in agreement with previous reports of reduced long bone growth and bone mass in fa/fa Zucker rats (37, 39, 50). Similar to leptin-resistant fa/fa rats with elevated fat mass and reduced whole-body BMC, however, fat mass is negatively associated with whole-body bone mass in children (28) and adolescent females (54). In addition, bones of obese or overweight children have been reported to fracture more often than those of children of normal weight, which suggests disproportionate bone mineralization, similar to the observations in the fa/fa rat (29, 30).

At 6 weeks of age, whole-body BA, BMC, and femur size were greater in the fa/fa genotype, which also had a 29% higher body weight. By 14 weeks of age, the difference in body weight between genotypes had increased to 67%. This was accompanied a 7% and 4% higher BMD in the femur and lumbar spine, respectively, in the fa/fa rats. This suggests that gains in fat mass may be positively associated with BMD in the femur and spine, which has been reported in overweight children (55). This is a novel finding, because femoral BMD in fa/fa rats has been reported previously to be lower or the same as that of lean rats (39, 50, 56, 57). The differences among studies may be explained by the older age (39, 56, 57) and female sex (39, 56) of rats. The study by Mollard et al. (50) had less statistical power and used a diet with10% lipid, explaining in part why the conclusions differ fromthose of the present study despite a similar trend in femoralBMD.

Bone size was reduced and regional bone mass elevated in the fa/fa rats in comparison to lean rats, but serum osteocalcin did not differ between genotypes, indicating that the fa/fa and lean rats had similar rates of bone formation and modeling. Therefore, the reduced femur size but elevated bone mass may result from differences in the bone structure and formation of cortical and trabecular bone. In addition, the higher femoral BMD was accompanied by a higher concentration of phosphorus, not calcium, in the femur, suggesting differences in the bone matrix structure between genotypes. Areas that are richer in trabecular bone in the femur were reduced in size in the fa/fa rats (proximal epiphysis and femur neck), which has been observed previously (50). Lower femoral trabecular bone thickness and greater cortical bone width in fa/fa rats also has been reported (37).

It is not surprising that CLA did not modify bone mass in the fa/fa or lean rats. In young and ovariectomized rats, 0.25%–1.0% (w/w) CLA isomers did not increase femur or humerus length, BMC, and BMD (7, 58, 59). The present study provides further evidence that bone mass may not be altered by CLA treatment. Bone modeling, however, which was not assessed in the diet groups of the present study, may be improved with intakes of CLA. Reduced bone resorption and prostaglandin E₂ synthesis by bone, increased intestinal calcium absorption, and greater expression of insulin-like growth factor–binding proteins in bone were observed in healthy and ovariectomized rodents fed 1% (w/w) CLA isomers (7, 13, 58). Over time, all three mechanisms may serve to increase bone mass, but to our knowledge, this has not been investigated in a long-term animal study. In humans, a 2-year CLA supplementation trial conducted in males reported a positive change in bone mineral mass between 1 and 2 years of supplementation, but after 2 years, bone mass did not differ significantly from baseline measurements (19). Thus, it seems that sustained CLA supplementationhas no negative effect on bone when fed in adulthood, but anylong-term changes in bone mass when CLA is fed during earlylife are presently unknown. From the present study as well asothers, however, it appears that during bone mineralizationand consolidation, no negative effects are observed in bone,but this may be dependent on the amount of CLA that is fed andon the n-6 to n-3 PUFA ratio in the diet.

The effects of CLA on bone mass have been shown to be dependent on the amount of CLA provided in the diet. Feeding less than 0.5% (w/w) CLA has been shown to have no effect, whereas feeding 0.5%–1.0% (w/w) had a positive effect on bone in mice, rats, and pigs (6, 9, 58). Providing 0.4%–1.1% (w/w) CLAisomers resulted in no differences in bone mass in the presentstudy, suggesting that the effects of CLA on bone are not dependenton the amount of CLA consumed by the male Zucker rat.

In the present study, soybean oil was used as the base lipid for all diets. Soybean oil has a n-6 to n-3 PUFA ratio that is moderately low (7:1) in comparison to those of other oils, such as corn (57:1), and to the ratio generally consumed in a Western diet, which is approximately 20:1 to 30:1 (60). Previous studies using a low ratio of n-6 to n-3 PUFA (~10:1 or less) have found minimal effects of CLA on bone mass (7, 12, 13). Therefore, the primary fat source in the present study may have limited the response in bone to CLA treatment. Also, the control diet group may have received a diet that marginally stimulated osteogenesis, because a low n-6 to n-3 PUFA ratio has been shown previously to be beneficial to bone mass (1). Thus, statisticaldifferences between control and CLA diet groups may have beenmore difficult to elucidate.

In conclusion, at 6 and 14 weeks of age, fa/fa rats have reduced femoral growth, most notably in areas of trabecular bone. If reduced growth is accompanied by lower mineralization in these areas, fracture risk in the hip joint may be increased with age in this animal model. The fa/fa rat also appears to have disproportionate bone growth for body size, but greater body weight elevates BMD in the femur and spine. Supplementation with CLA did not benefit bone growth or whole-body mineralization in the fa/fa rats in comparison to lean rats. Further investigationis required to clarify the dietary conditions in which CLA maybenefit bone mass.

 

 

 

View this table: [in this window] [in a new window] Table 1. Lipid Composition of Diets (g/kg diet)

 

View this table: [in this window] [in a new window] Table 2. Whole Body, Femur, and Spine Lumbar Vertebrae Measures for Lean and fa/fa Zucker Rats at 6 or 14 Weeks of Agea

 

View larger version (22K): [in this window] [in a new window] Figure 1. Total feed intake (kg) over the 8-week study duration. Values are presented as the mean ± SEM (n = 10 rats/group). Differences among dietary groups were identified using least squares means testing. Bars with a different letter indicates a significant difference between groups (P < 0.05). Diet groups are as follows: Group 1, 0.4% (w/w) c9, t11 CLA; Group 2, 0.4% (w/w) t10, c12 CLA; Group 3, 0.4% (w/w) c9, t11 CLA and 0.4% (w/w) t10, c12 CLA; Group 4, 0.4% (w/w) c9, t11 CLA, 0.4% (w/w) t10, c12 CLA, and traces of other isomers; and Group 5, 0.4% (w/w) c9, t11 CLA, 0.4% (w/w) t10, c12 CLA, and 0.3% (w/w) other CLA isomers.

 

View larger version (26K): [in this window] [in a new window] Figure 2. Adjusted whole-body BMC after 8 weeks of dietary treatment with c9, t11 or t10, c12 CLA isomers. Values are presented as the mean ± SEM (n = 10 rats/group). Differences among dietary groups were identified using least squares means testing. Bars with a different letter indicates a significant difference between groups (P < 0.05). Diet groups are as follows: Group 1, 0.4% (w/w) c9, t11 CLA; Group 2, 0.4% (w/w) t10, c12 CLA; Group 3, 0.4% (w/w) c9, t11 CLA and 0.4% (w/w) t10, c12 CLA; Group 4, 0.4% (w/w) c9, t11 CLA, 0.4% (w/w) t10, c12 CLA, and traces of other isomers; Group 5, 0.4% (w/w) c9, t11 CLA, 0.4% (w/w) t10, c12 CLA, and 0.3% (w/w) other CLA isomers and control diet.

	Acknowledgments

 

We thank Danielle Defries, Natasha Ryz, Dielle Herchak, RobDiakiw, Jennifer Zahradka, and the staff at the University ofManitoba Fort Garry Campus Animal Care Facility for assistingwith animal feeding and care. We also thank the Manitoba Instituteof Child Health for use of the DXA equipment and Shirley Fitzpatrick-Wongfor assistance with the femur analysis. The kind gift of theCLA supplement from Bioriginal Food and Science Corporation(Saskatoon, Saskatchewan) is acknowledged.

	Footnotes

This research was supported by the Dairy Farmers of Canada andthe Natural Sciences and Engineering Research Council of Canada.This paper was presented in part at the Canadian Federationof Biological Sciences Conference, June 2005, in Guelph, Ontario,Canada (Burr LL, Taylor CG, Weiler HA. Femur bone mass, butnot whole body bone mass, is greater in fa/fa Zucker rats thanin lean Zucker rats. CFBS Proc 48:83, 2005.

Received for publication February 17, 2006.

Accepted for publication April 24, 2006.

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