Kidney International, Vol. 48 (1995), pp. 475—480

Flaxseed: A potential treatment for lupus nephritis

WILLIAM F. CLARK, ANWAR PARBTANI, MURRAY W. HUFF, EVELYN SPANNER, HELEN DE SAUS,IAN CHIN-YEE, DIANA J. PHILBRICK, and BRUCE J. HOLUB

Department of Medicine, University of Western Ontario, London; Department of Nutritional Sciences, University of Guelph, Guelph; and Nutrition &Kidney Disease Research Group, Victoria Hospital Research Institute, London, Ontario, Canada

Flaxseed: A potential treatment for lupus nephritis. Flaxseed is rich ina-linolenic acid (a-LA) which has anti-atherogenic properties, and lignanswhich are platelet activating factor (PAF)-receptor antagonists. Theseconstituents of fiaxseed, and its beneficial effects in the MRL/lpr lupusmouse prompted us to perform this dosing study in lupus nephritispatients. Nine patients were enrolled, eight of whom completed the study.After the baseline studies, patients were given 15, 30, and 45 g offlaxseed/day sequentially at four week intervals, followed by a five-weekwashout period. Compliance, disease activity, blood pressure, plasmalipids, rheology, PAF-induced platelet aggregation, renal function, andserum immunology were assessed. Flaxseed-sachet count and a significantincrease of serum a-LA indicated good compliance for 15 and 30 g doses.Total and LDL cholesterol, and blood viscosity were significantly reducedwith 30 g and to a lesser extent 45 g doses. PAF-induced plateletaggregation was inhibited by all doses. There was a significant decline inserum creatinine with 30 and 45 g, and a concomitant increase increatinine clearance with increasing flaxseed dose. Proteinuria was re-duced with 30 g and to a lesser extent with 45 g of flaxseed. ComplementC3 was significantly elevated by all three doses. CD11b expression onneutrophils, a measure of C3bi receptors, was significantly reduced withthe 30 g dose. In conclusion, 30 g flaxseed/day was well tolerated andconferred benefit in terms of renal function as well as inflammatory andatherogenic mechanisms important in the pathogenesis of lupus nephritis.

Nutritional intervention in progressive kidney disease is under-going a renaissance [1—5]. Our interest in omega-3 fatty acids ledus to carry out this current dosing study of flaxseed in patientswith lupus nephritis [6—10]. Flaxseed is a rich source of a-linolenicacid (a-LA, 8:3n3). However, it is not only the anti-mitogenic andlipid lowering qualities of the a-LA, but also the lignan contentwith its potent PAF receptor antagonist properties that attractedour interest [11—14]. PAF is a major participant in the activationand propagation of the inflammatory reaction, and is known to beelevated in patients and animals with lupus nephritis. PAFinhibition in lupus animal models is associated with increasedsurvival and decreased proteinuria [15—18]. We recently reportedthe effects of a 15% flaxseed diet in the MRLI1pr mouse model oflupus nephritis [8]. The MRLI1pr mice spontaneously develop alymphoproliferative syndrome associated with a progressive sys-temic autoimmune disease involving kidney, heart, skin and jointssimilar to severe forms of human lupus nephritis [19]. The

Received for publication December 8, 1994and in revised form March 1, 1995Accepted for publication March 1, 1995

© 1995 by the International Society of Nephrology

flaxseed significantly preserved GFR, reduced proteinuria, lym-phoproliferation and mortality in the MRLIIpr model and wasmuch more potent than previous studies with fish oil [8]. We alsostudied the effects of flaxseed versus its constituents (flax oil anddefatted flax) in the MRLI1pr mice, and observed that the wholeflaxseed conferred superior benefit compared to its oil or defattedconstituents [9]. These encouraging results prompted us to inves-tigate the short term effects of three different doses of flaxseed onimmune, inflammatory, rheologic, platelet and lipid parameters inlupus nephritis. We wished to determine a dose of flaxseed whichwas well tolerated and would have a significant effect on patho-genic mechanisms known to be involved in this progressivescarring disorder.

Methods

Patients

Nine patients were recruited for this study. All patients met theminimum American Rheumatism Association's criteria for thediagnosis of systemic lupus erythematosus (SLE) and all had adocumented history of positive ANA, and at least one episode ofelevated anti-DNA binding with proteinuria > 1 g124 hours, redcells and red cell casts on urinalysis [20]. Seven of the ninepatients also had undergone renal biopsy (Table 1). Eight patientswere receiving prednisone in doses ranging from 5 mg on alter-nate days to 10 mg daily, and two were receiving Imuran.Additional characteristics of the patients are provided in Table 1.

Trial design

The study was divided into four phases. The first three phaseswere of four weeks duration and the last phase was of five weeksduration. In phase 1 the subjects ingested 15 g of flaxseed dailywith cereal, tomato or orange juice and this was increased to 15 gtwice daily in the ensuing four weeks. In the final four weeks ofstudy the flaxseed was increased to 15 g thrice daily, and this wasfollowed by a five-week washout period.

Dietary intake assessment

A three-day dietary intake record was obtained from eachpatient at each phase of the study and analyzed using theCanadian Nutrient File Database (Nutrition Res. Div., HealthProtection Branch, Ottawa, ON, Canada). Total calories, protein,fat, cholesterol, saturated fatty acids, monounsaturated fatty acidsand polyunsaturated fatty acids were calculated. In addition,protein catabolic rate (PCR glday) was calculated from protein

475

476 Clark et a!: Flasseed in lupus nephritis

Table 1. Characteristics of the patients who completed the study

Patientnumber

Ageyears Gender

Lengthof lupusmonths

Renalbiopsy

classification

1 36 female 96 DPGN2 43 female 8 FPGN3 32 female 36 DPGN4 35 female 122 DPGN5 51 female 84 DPGN6 72 male 84 DPGN7 40 male 84 DPGN8 47 female 36 DPGN

Mean age was 44.5 years; mean length with lupus was 68.8 months.Biopsy classifications: are DPGN, diffuse proliferative; FPGN, focal

proliferative.

excretion, urea excretion and urea generation determinationaccording to Sargent and Gotch [21].

ComplianceSachets containing 15 g of flaxseed were provided to each

patient. Extra sachets were given for each phase of the study.Sachets returned at the end of each phase provided a measure offlaxseed intake. However, the actual ingestion of flaxseed wasassessed from the relative increase of a-linolenic acid from thefatty acid analyses of serum phospholipids.

Fatly acid contents of serum phospholipidsSerum phospholipids were extracted and assessed for fatty acids

as previously described [22]. In brief, the lipid extracts weresubjected to thin layer chromatography. The phospholipid bandscorresponding to different lipid fractions were isolated and as-sessed for fatty acid composition using gas chromatography.

SLE Disease Activity Index

At each visit patients were assessed by the same physician whoscored the SLE Disease Activity Index (SLEDAI) [23].

Blood pressure

Supine and standing blood pressure was obtained by a mercurysphygmomanometer and taking Korotkoff's fifth sound as thediastolic blood pressure cut-off.

Plasma lipidsFasting blood samples (12 to 14 hr) were collected in EDTA on

ice. Plasma was assayed for total triglycerides, total cholesterol,HDL-, LDL-, and VLDL-cholesterol as previously described [22].All assays were standardized to plasma secondary standards forwhich the target values were assigned at the Lipid Research ClinicLaboratory, University of Toronto (St. Michael's Hospital, To-ronto, ON, Canada).

RheologyThe viscosity of heparinized whole blood relative to water was

measured using a white cell pipette at room temperature accord-ing to the method of Wright and Jenkins [24]. For red cellflexibility (RCF) heparinized blood was centrifuged for twominutes in a Clay Adams Autocrit centrifuge (Clay Adams Co.,Parsippany, NJ, USA) at 200 X g. The red cell flexibility was

measured as a percentage of cell packing/mm as outlined by Sirs[25].

PAF-induced platelet aggregation

Platelet rich plasma was prepared from citrated blood andplatelet count was adjusted to 250,000/1.d using homologousplatelet poor plasma. Platelet aggregation was performed aspreviously described [22]. The threshold aggregation concentra-tion (TAC) for PAF (AcGEPC; Sigma Chemical Co., St. Louis,MO, USA), defined as the minimum amount of PAF required foran irreversible aggregation within five minutes, was obtained.

Renal function tests

Urinary proteins were measured using a routine sulfosalicylicacid precipitation method. Serum creatinine was measured bykinetic Jaffé chromogen reaction. Creatinine clearance was calcu-fated using the formula: urine creatinine (mol1liter) X urinevolume (ml/min)/serum creatinine (mol/liter). Serum urea wasmeasured by urease-enzymatic method using Paramax AnalyticalSystem (Baxter, Irvine, CA, USA).

Serum immunology

Serum complement (C3 and C4) were measured by scatteredlight turbidity in a nephelometer (BNA Nephelometer, Behring-werke AG Diagnostica, Marburg, Germany). The total hemolyticcomplement (CH1) was measured by the antibody-coated eryth-rocyte hemolytic method and expressed as lytic units of comple-ment. Antibodies to double-stranded DNA antibodies (ds-DNAbinding activity) were measured in sera using the Farr ammoniumsulphate precipitation technique.

Flow cytometly studies

Lymphocyte subsets were determined using single, dual andthree-color monoclonal antibody (MoAb) combinations. Thefollowing MoAb were used: CD3, CD4, CD8, CD14, CD19,CD25, CD29, CD45 (Coulter Electronics, Miami, FL, USA),CD5, CD 16, CD56 (Becton Dickinson, Mountainview, CA, USA),and CD11b, which recognizes C3bi receptors (AMAC Inc., West-brook, ME, USA). Whole blood samples were incubated withMoAbs for 30 minutes at 4°C. The red cells were then lysed usingImmunoprep reagents (Coulter Electronics), washed in phos-phate buffered saline containing 1% bovine serum albumin,resuspended and analyzed by flow-cytometry (Coulter Electron-ics). Lymphocyte and neutrophil populations were identified bylight scatter characteristics (side scatter vs. forward angle lightscatter). Log fluorescence data were collected in single and dualparameter histograms using the standard instrument filter config-uration and analysis package (positive regions and quad stat).

Statistical analysis

Serial data for each patient from baseline through to the postfive-week washout period enabled the use of repeated measuresanalysis of variance (ANOVA), which if significant was followedby Bonferroni t-tests to discern the sources for the statisticaldifferences. A value of P � 0.05 was regarded statistically signif-icant. The data in the manuscript represent mean SEM.

Clark et al: Flaxseed in lupus nephritis 477

Table 2 Dietaiy fat and protein intake

4 Weeks 4 Weeks 4 Weekspost-15 g post-30 g post-45 g 5 Weeksflaxseed/ flaxseedl flaxseed/ of washout

Baseline day day day period

Fat intake 72 6 86 14 85 8 80 7 60 5

gidayProtein intake 63 9 67 7 74 6 65 6 56 7

giday fromdieta,y records

PCRg/day 70±9 62±8 66±8 70±8 60±9based onurea kinetics

The values include protein (25%) and fat (40%) contributions fromflaxseed actually consumed.

There was no significant difference in intake for any of the dietarycomponents over the study period.

There was no significant difference between protein intake based ondietary records vs. PCR based on urea kinetics.

Results

Patients

Eight of nine patients completed the study. One dropped outafter the first four weeks of the study due to scheduling difficultiesat his work place. Three subjects experienced difficulty ingestingthe 15 g flaxseed three times daily (45 g total dose) due toincreased laxation. All patients tolerated well the 15 g and 30 gflaxseed dosage, either mixing it with orange or tomato juice or asa cereal topping. No patient required a change in their medica-tions during the 17 weeks of the study.

Dietary intake assessment

The three-day dietary intake records showed no change in theintake of total calories, protein, fat, cholesterol, and saturated,monounsaturated and polyunsaturated fatty acids. The PCRcalculations based on urea generation confirmed no change forprotein intake determined from the dietary records. Table 2provides the data for fat, protein intake, and PCR (glday).

ComplianceCompliance was excellent for the flaxseed dosage of 15 g/day

(15 1 g) and 30 g/day (31 3 g). However, at the 45 g/daydosage the measurement was 43 10 g, indicating some variabil-ity as evidenced by three of the patients reporting difficulty incomplying with the 45 glday dose due to increased taxation. Thispattern of compliance was confirmed by the increase of thea-linolenic acid levels in the serum phospholipid from the base-line to four-weeks post-15 g flaxseed, followed by a statisticallysignificant increase with the 30 g/day dosage, and a moderatedecline at the 45 g/day dosage, with a return to baseline value afterthe five-week washout period (Table 3). Eicosapentenoic acid(EPA) as well as the ratio of n3/n6 fatty acids also increased withall of the flaxseed doses; however, the linoleic acid and thearachidonic acid levels were unchanged (Table 3), which is inaccord with the subjects not altering their diets while taking theflaxseed.

SLE Disease Activity Index

The SLEDAI scores ranged from 9 to 11 and were unchangedthroughout the study.

Blood pressure

The mean blood pressure was unchanged throughout all phasesof the study (Table 4). None of the patients were on, or requiredany anti-hypertensive treatment.

Plasma lipids

The flaxseed exerted a significant cholesterol lowering effect at30 glday, reducing the total cholesterol by 11% and the LDLcholesterol by 12% (Table 5). This effect was sustained over thefour weeks of 45 g/day dose (9% for total and 10% for LDLcholesterol) and the five-week washout period (7% for total and11% for LDL cholesterol), indicating a prolonged lipid loweringeffect.

Rheology

Whole blood viscosity (WBV) was significantly reduced from abaseline of 3.03 0.14 to 2.47 0.07 at the 30 g/day dose level.This reduction in WBV was significantly maintained following thefive-week washout period at 2.52 0.11, indicating that theflaxseed was still exerting an effect. Red cell flexibility increasedwith the 30 g/day flaxseed (20.1 1.9) but this did not achievestatistical significance compared to baseline value (19.7 1.7).

Platelet aggregation to PAF

The TAC of PAF was increased with 15 g (35.9 5.5 tg), 30 g(27.1 7.4 g) or 45 g (25.4 9.3 g) of flaxseed compared tobaseline value of 16.1 6.4 g PAF. At the end of the five-weekwashout period the TAC approximated the baseline value (17.95.9 pg).

Renal function tests

Serum creatinine was significantly reduced with the 30 and 45day flaxseed dose with a return towards pre-treatment valuesfollowing the five-week washout period. The creatinine clearancereflected this improvement, rising with increasing flaxseed doses(Table 4). The serum urea were unchanged. Urinary proteindeclined with 15 g and 30 g, and rose slightly with 45 g dose, butreturned to baseline level post-washout period (Table 4). Threepatients with the baseline proteinuria of > 1 g/24 hr showedmarked reduction, particularly by the end of the 30 g/day dose(from 1.2, 2.5, 6.6 to 0.4, 1.8, 1.1 g/24 hr, respectively).

Serum immunology

Complement C4 and anti-DNA antibody titer were unchangedthroughout the study period. There was a trend towards a rise inC3 level during flaxseed dosing, with a significant decline follow-ing five-weeks of washout period (Table 6). CH100 levels rose with45 g of flaxseed which persisted through the washout period.

Flow cytometty studies

No significant alterations were observed with flaxseed dosing intotal T helper (CD3+/CD4+), helper inducer (CD4+/CD29+),suppressor inducer (CD4+/CD45RA+) subsets, natural killercells (CD3-/CD16+ and/or CD56+) or cytotoxic T cells (CD3+/CD16+ and/or CD56+). There was a reduction in CD11b expres-sion on neutrophils based on mean channel fluorescence (MCF)during the 30 g/day flaxseed dosing (6.5 0.6) compared tobaseline (11.5 1.9) and the post-five-week washout period (13.5

2.3).

Baseline

Linoleic acid 16.50 0.81(18:2n6)

Arachidonic acid 9.53 0.44(20:4n6)

a-Linolenic acid 0.18 0.02(18:3n3)

Eicosapentaenoic 0.93 0.08acid (20:5n3)

Docosapentaenoic 0.72 0.07acid (22:5n3)

Docosahexaenoic 3.18 0.21acid (22:6n3)

Ratio: 0.17 0.01n3/n6 fatty acids

a Significantly different from baselineb

Significantly different from 5 week post-washout period

Mean blood pressuremmHg

Proteinuriamg124 hr

Serum creatininepinol/liter

Creatinine clearancemi/mm

Serum ureammol/liter

Only standing BP is given, since there was no difference between standing and supine BP.Proteinuria was non-normally distributed; geometric means and ranges in the parentheses are given.For serum creatinine, creatinine clearance and serum urea data represent mean SCM.a Significantly different from baseline

4 Weekspost-15 g

fiaxseed/day

6.23 0.43

1.56 0.22

3.83 0.43

0.84 0.10

1.76 0.22

4 Weekspost-45 g

flaxseed/day

5.79 0.8a

1.48 0.23

3.48 0.37

0.81 0.15

1.56 0.23

5 Weeksof washout

period

5.90 0.40

1.52 0.21

3.45 0.39

0.91 0.10

1.48 0.24

Discussion

Systemic lupus erythematosus is an autoimmune disease with abi-modal mortality pattern punctuated by early inflammatory andlate atherosclerotic events [26—28]. The potential of flaxseed toalter immune, inflammatory and atherosclerotic events coupledwith our findings in murine lupus [8, 9] led us to construct a dosing

study in patients with lupus nephritis. This study provides onlypreliminary evidence for an effect of flaxseed on the diseaseprocess since the duration is short and patients are used as theirown controls.

Flaxseed was well tolerated at 15 and 30 g/day, and this wascorroborated by compliance measures with sachet counts as well

478 Clark et al: Flaxseed in lupus nephritis

Table 3. Select fatty acid contents of serum phopsholipids (mean SCM)

4 Weeks 4 Weekspost-is g post-30 g

____________________________________ flaxseed/day fiaxseed/day

16.81 0.67 16.11 0.25

8.96 0.75 9.03 0.33

0.25 0.04 0.38 004ab

1.01 0.08 1.28 022b

0.79 0.05 0.82 0.07

3.17 0.14 3.22 0.01

0.25 0.02a 0.28 002a,b

4 Weekspost-45 g

flaxseed/day

16.59 0.57

9.18 0.43

0.30 0.03

1.25 008b

0.93 0.07

3.51 0.16

0.25 0.ola

5 Weeksof washout

period

17.12 0.53

8.66 0.51

0.21 0.04

0.80 0.08

0.69 0.06

3.27 0.15

0.21 0.01

Baseline

109 4

657(154; 2810)112 15

81 18

6.78 0.92

Table 4. Blood pressure and renal physiology

4 Weeks 4 Weekspost-15 g post-30 g

fiaxseed/day fiaxseed/day

105±4 106±4

578 462(189; 1764) (170; 1256)98±14 96±12a

90±20 90±18

7.21 0.98 7.78 1.12

4 Weekspost-45 g

fiaxseed/day

105 3

503(147; 1722)90 13101 23

7.35 1.09

5 Weeksof washout period

109 4

663(142; 3098)104 17

92 15

7.25 1.59

Baseline

Total cholesterol 6.37 0.45mmoi/liter

HDL-cholesterol 1.58 0.24mmol/liter

LDL-cholesterol 3.88 0.41mmol/iiter

VLDL-cholesterol 0.93 0.14mmoi/liter

Triglycerides 1.62 0.25mmoi/iiter

a Significantly different from baseline

Table 5. Plasma lipids (mean SEM)

4 Weekspost-30 g

____________ fiaxseed/day

5.64 0.36a

1.51 0.26

3.42 0.35

0.71 0.11

1.58 0.25

Clark et al: Flaxseed in lupus nep/iritis 479

Table 6. Serum immunology (mean 5EM)

4 Weeks 4 Weeks 4 Weekspost-iS g post-30 g post-45 g 5 Weeksflaxseed/ flaxseed/ flaxseed/ of washout

Baseline day day day period

C3 610 69 657 8&' 656±61" 649 73" 560±65mg/liter

C4 196±36 205±40 202±34 213±42 182±33mg/liter

CH100 83±12 90±15 98±17 63±14a 67±o9aU/mI

DNA-b 125±42 137±45 140±39 133±37 123±38lU/mi

a Significantly different from post-30 g flaxseed/day"Significantly different from 5 weeks post-washout

as the increase of a-linolenic acid levels in serum phospholipids.However, the 45 g/day dose of fiaxseed was not well tolerated dueto increased laxation and was associated with increased variabilityin compliance as well as a decrease in a-linolenic acid levelscompared to the 30 glday dose. Dietary enquiry and the proteincatabolic rate (PCR) determination indicated that patients did notalter their diet, particularly protein intake, during the study period(Table 2), which was in keeping with the constancy of seriallinoleic and arachidonic acid levels (Table 3).

Total and LDL cholesterol levels were significantly decreasedwith the 30 and to a lesser extent with 45g,'day flaxseed, and thiseffect persisted after the five week washout. The lipid loweringeffect of fiaxseed was not associated with a change in total fat,cholesterol or saturated fatty acid intake based on the dietaryenquiry. The fiaxseed is composed of 38 to 40% fat of which 50%is a-linolenic acid, and 26 to 33% dietary fiber and mucilage[29—31j. Thus, it is not clear whether either, or both, are respon-sible for the significant reduction in LDL cholesterol levels.Hypercholesterolemia is a major feature of the dyslipidemia ofSLE [32]. The marked hypocholesterolemic effects of the flaxseedmay reduce not only the accelerated atheromatous vasculardisease but also the renal tissue scarring in patients with lupusnephritis [33—35]. The observed lipid lowering effects, coupledwith the significant reduction in whole blood viscosity with 30glday fiaxseed, suggests the potential for a major reduction in theincidence of coronary artery disease and stroke in this high riskpatient population [26—28, 36]. In this regard it is important tonote that an increased intake of a-linolenic acid-rich diet, and theresultant increase in a-linolenic acid, EPA and n3/n6 ratio inserum phospholipids (also observed in the present study; Table 3)significantly decreased cardiac death and the non-fatal myocardialre-infarction in patients with coronary heart disease [37].

Renal function, assessed by serum creatinine, creatinine clear-ance and urinary protein measurements, was surprisingly alteredin the short duration of the study. The significant decline in serumcreatinine coupled with an increase in creatinine clearance, and adecrease in proteinuria does not suggest a pharmacologic actionof the flaxseed on renal hemodynamics. Theoretically, this findingis more compatible with a direct alteration in glomerular base-ment membrane properties which would be associated withincreased filtration and improved permselectivity [38]. The reduc-tion in serum viscosity which persisted after five weeks of thewashout period is similar to our findings with fish oil [6]. Thereduction in whole blood viscosity could potentially contribute to

a decrease in glomerular capillary permeability and the conse-quent reduction in proteinuria [39}.

The flaxseed ingestion was associated with an increase in theamount of PAF required to cause platelet aggregation and thiseffect disappeared following the five week washout. We hadpreviously documented the ability of flaxseed to inhibit PAF-induced platelet aggregation in mice [8]. Flaxseed is the richestnatural source of lignans, and produces 75- to 800-fold moremammalian lignans than 66 other plant foods in vegetarian diets[40]. Lignans are a group of substances containing a dibenzylbutane skeleton that produces specific reversible and competitiveinhibition of PAF [13].

The fiaxseed dietary supplementation did not alter the SLEDAIin the subjects nor the mean blood pressure, which is alsocompatible with our findings in previous studies of omega-3 fattyacids (fish oil) in lupus nephritis [6, 7]. We observed rising levelsof C3 relative to the flaxseed dose. The reduction in CD11bexpression (a measure of C3bi receptors) on the neutrophil with30 glday dose of fiaxseed was also noted. Other immune serologicparameters, as well as T cell markers were largely unchanged bythe flaxseed dosing. Flaxseed oil has been shown in 10 healthyvolunteers to suppress the proliferation of peripheral mononu-clear cells when stimulated by phytohemagglutinin or concanavo-lin A in the delayed hypersensitivity response to seven recallantigens [11]. However, the fiaxseed oil did not produce a changein the number of helper, suppressor or total T and B cells inperipheral blood in that study [11], which is in keeping with ourflow cytometry data.

We conclude that 30 g/day of fiaxseed is well tolerated bypatients with SLE nephritis, and it exerts significant effects onrenal function, plasma lipids, blood viscosity and complement C3levels. These preliminary findings of the dosing study are ofinterest and may serve to stimulate larger control studies toconfirm or refute fiaxseed's potential as a treatment for patientswith lupus nephritis.

Acknowledgments

A portion of this study was presented at the 27th annual meeting of theAmerican Society of Nephrology at Orlando in October, 1994 (Abstract#119P, JASN 5:348, 1994). We acknowledge the support of the KidneyFoundation of Canada, Victoria Hospital Research Institute and Depart-ment of Medicine, Victoria Hospital, London, Ontario. We thank DennisMcIntosh, Omega Life Products Ltd., Melfort, Saskatchewan, for provid-ing fiaxseed, Esmé French and Sharon Clark for study coordination, andTeresita Daite, Frances Andrus, Sandi Kleintsivcr, Lori Rudzitis, andMichael Keeney for technical assistance.

Reprint requests to Dr. WF. Clark, Department of Medicine, VictoriaHospital, London, Ontario, Canada, N6A 4G5.

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