High density lipoproteins during hypolipidemic therapy

215

Atherosclerosis, 35 (1980) 215-228 Scientific Publishers, 0 Elsevier/North-Holland

HIGH DENSITY A Comparative

LIPOPROTEINS

Ltd.

DURING

HYPOLIPIDEMIC

THERAPY

Study of Four Drugs

MARIAN C. CHEUNG, HAZZARD

JOHN J. AJ,BERS,

PATRICIA

W. WAHL and WILLIAM

Northwest Lipid Research Clinic, Harborview Medical Center, Department Department of Biostatistics, University of Washington, Seattle, WA 98104 (Received 26 February, 1979) (Revised, received 15 October, (Accepted 17 October, 1979)

R.

of Medicine and (U.S.A.)

1979)

Summary The high density lipoprotein (HDL) response of 14 hyperlipidemic subjects to four hypolipidemic agents was studied through serial measurement of HDL cholesterol and apolipoproteins A-I and A-II before and during 3 months each (separated by 2 months off drug) of clofibrate (2 g/day, n = 14), colestipol (20 g/day, n = 12), para-amino salicylic acidascorbate (PAS-C, 6-8 g/day, n = 14) taken in random sequence and oxandrolone (7.5 mg/day , n = 11) as the final drug. The maximal effect of each drug appeared by the first monthly evaluation, and A-l, A-II and HDL cholesterol levels returned to pretreatment levels by one month after discontinuation of each agent. With clofibrate, HDL cholesterol increased by 16 + 20% from baseline (mean f SD) (P < 0.05), A-I by 11 + 13% (P < 0.05) and A-II by 39 -L 17% (P < 0.01). During oxandrolone HDL cholesterol declined by 36 + 20% from baseline (P < O.Ol), A-I by 21 It 13% (P < O.Ol), and A-II by 16 _+11% (P < 0.025). Neither PAS-C nor colestipol exerted major effects on HDL, or any of the variables although both were associated with a slight rise in the A-I/A-II ratio (11 + 15% and 12 f 12%, respectively). Key words:

Genetic hyperlipemia

-High

density lipoprotein - Hypolipidemic

therapy

These studies were supported by contract NIHV 12157A from the National Institutes of Health, Lipid Metabolism Branch, grant HL-22285. the American Heart Association of Washington, the Ayerst, HeIIwig and Upjohn Pharmaceutical companies, and the Clinical Research Center of Harborview Medical Center, supported by NIH grant RR-37. Address reprint requests to Dr. William R. Hazzard, Northwest Lipid Research Clinic, 325 Ninth Avenue, Seattle, WA 98104. U.S.A.

216

Introduction High density lipoproteins (HDL) have recently received great attention because of their putative value as a protective (i.e., “inverse risk”) factor against atherosclerotic cardiovascular disease. This concept, clearly in evidence in publications of a quarter-century ago [l-3] has received contemporary support from population studies [ 4-61, clinical investigation and cell research [ 7,8]. Hence, current evaluation of any mode of lipidemic intervention should take into consideration its separate and interrelated effects upon HDL, low density lipoproteins (LDL), and very low density lipoproteins (VLDL) in computing its potential value in reducing cardiovascular risk. Because of the relative ease and widespread availability of lipid measurements, the levels of these lipoproteins have been conventionally estimated in terms of their lipid (especially cholesterol) content. However, this approach has certain limitations. For instance, although population studies have disclosed that plasma triglyceride and HDL cholesterol levels are inversely related [ 5,6,9] there is no evidence showing that plasma triglyceride regulates HDL concentrations. A recent study from this laboratory in fact showed that in hypertriglyceridemic myocardial infarction survivers, low levels of HDL cholesterol could not be attributable to hypertriglyceridemia alone, but may also be attributable to a low level of the HDL apolipoproteins (A-I and A-II) [lo]. Moreover, HDL density subclasses, which may have different implications for cardiovascular risk [ 111, differ in their relative proportions of A-I and A-II [ 121. Thus, alterations in the A-I/A-II ratio suggest changes in the HDL subclass distribution. Hence, measurements of A-I and A-II levels may provide additional information in estimating the potential effects of lipidemic intervention over and above that afforded by measurements of lipoprotein lipids alone. In addition to dietary advice, current hypolipidemic regimens include a variety of drugs of widely differing (and often poorly understood) mechanisms. To date, few studies of such agents have systematically evaluated their effects upon HDL apolipoprotein levels and composition. In this study, we have attempted to redress this deficiency through serial measurements of HDL cholesterol, A-I and A-II levels in hyperlipidemic subjects who ingested up to 4 different hypolipidemic agents for 3 months each (separated by 2 months off drug) in which the order of administration is randomized (except for oxandrolone, which was the final drug in each instance). The results suggest important effects on HDL cholesterol and apolipoprotein levels for 2 of these drugs, clofibrate (Atromid@) and oxandrolone (Anavar@). Methods

Subjects Volunteers were recruited from among subjects referred to the Northwest Lipid Research Clinic. After the principles and requirements of the study were explained in detail, informed consent was obtained; this included withholding from the volunteer knowledge of his or her lipid levels until completion of the study. Nine of the fourteen participants were. males and ranged in age from 28 to 66; the average age was 41 years. A detailed family study was then initiated

as described [ 131. This permitted classification of the hyperlipidemia into one of 4 inherited disorders in 10 of 14 cases: 5 (including a mother and son) had familial hypercholesterolemia, 3 had type-III hyperlipoproteinemia, and 1 each had familial combined hyperlipidemia and familial hypertriglyceridemia. All subjects with familial hypercholesterolemia had tendinous xanthomas. The diagnosis of type-III hyperlipoproteinemia required the repeated demonstration of beta-VLDL on agarose electrophoresis [ 141 of the isolated d < 1.006 lipoproteins and a ratio of VLDL cholesterol: plasma triglyceride ratio consistently exceeding 0.30 [ 151. In addition, 2 of the 3 participants with this disorder have subsequently been demonstrated to lack isoapolipoprotein E3 [16]. Family studies on the remaining 4 participants did not permit a genetic classification. Of note, all 4 tended to have combined hyperlipidemia (i.e., elevations of both cholesterol and triglyceride). Two were twins, purportedly monozygotic. Experimental design and clinical aspects of this study All studies were performed on an ambulatory basis at the Clinical Research Center at Harbor-view Medical Center. These outpatient visits occurred at monthly intervals, 22 in number, for subjects completing the entire 4drug sequence. Three years were required for the entire study. At the first visit, a complete cardiovascular history and physical evaluation were obtained, including resting and near-maximal exercise electrocardiography. At the same visit, the subjects were instructed in a diet moderately restricted in cholesterol (ca. 400 mg daily) and saturated fat (polyunsaturated/saturated ratio 0.6-1.0). Thereafter, the diet was reinforced by a review by a registered dietitian at each monthly visit, emphasis being placed upon consistency of dietary pattern. Other monthly data gathered by the research nurse included a form-standardized interval medical history with special reference to possible drug side effects. After 3 such baseline visits, the first drug was dispensed by the nurse after instruction in its potential side effects by a clinical investigator, who thereafter adjusted drug dosage as necessary to control side effects. All unused drug was returned each month and adherence calculated by a pill or packet count. No independent method of assessing drug adherence was employed. Each drug i.e., clofibrate (2 g/day) colestipol (20 g/day), para-amino salicylic acid (6-8 g/ day), and oxandrolone (7.5 mg/day), was taken for a 3-month period, followed by 2 months without hypolipidemic drug therapy before the next agent was begun. Subjects were randomly allocated to one of three drug sequence groups determined by an experimental model designed to balance the order in which clofibrate, colestipol, and PAS-C were given among the 3 groups. Oxandrolone was the final drug taken in every case in which it was tested. Plasma samples All blood samples were drawn from an antecubital vein after an overnight (12-14 h) fast into Vacutainer@ tubes containing disodium EDTA, 1 mg/dl, according to Lipid Research Clinics’ protocols [ 171. Cells were removed by low-speed centrifugation at 4°C within 2 h. Plasma samples for apoprotein analyses were stored at -20°C with 0.05% (wt/vol) sodium azide in sealed Wheaton vials until the day of analysis. Aliquots for lipid analyses were stored at 4°C and processed within 72 h.

218

Lipid analysis Cholesterol and triglyceride were measured by AutoAnalyzer II techniques [17,18] in whole plasma and plasma fractions. HDL was determined by heparin-manganese precipitation of the apolipoprotein-B containing lipoproteins (VLDL and LDL) from whole plasma or the d > 1.006 fraction [17]. For cholesterol analysis, the coefficient of variation was less than 4% and accuracy within 5% of the target value; for glyceride analysis, the coefficient of variation was less than 5% and accuracy within 10%. Immunoassay procedures Plasma apolipoproteins A-I and A-II were determined by radial immunodiffusion assays, as previously described [9,12]. A-I and A-II were measured with a coefficient of variation of less than 5%. To avoid between-assay variation, all samples from a given subject were assayed on the same plate. Statistical analysis Differences in apolipoprotein, HDL cholesterol and adherence among treatments were statistically analyzed by analysis of variance techniques. A twofactor design for repeated measures [19] was employed to test for drug treatment and sequence effects and any interaction between the drugs and their order of administration. Due to unequal sample sizes in the 4 treatments, a least squares solution for the effects and sums of squares was used. Mean differences between each drug and its own prior control were compared to the within subjects’ experimental error variance in order to statistically determine which drugs significantly altered HDL cholesterol and apolipoprotein values. The probability levels for each test were adjusted to guard against the risk of multiple testing. Pearson’s correlation coefficient was used to describe the linear association between apolipoproteins and lipids; log triglyceride values were used in these calculations. Results Descriptive statistics for plasma A-I, A-II, HDL cholesterol, cholesterol and triglyceride are given in Table 1 for the baseline period and each treatment and its control period. The heterogeneity of the subject group is reflected in the large standard deviations within each variable at baseline. Although mean levels of A-I, A-II, and HDL cholesterol were not substantially lower than the respective means in a healthy employee population [ 121, this was clearly attributable to the presence in this group of two women with hyperalphalipoproteinemia (HDL = 93, 74; A-l = 170, 212; A-II = 43,33, respectively) and familial hypercholesterolemia. The expected inverse correlation between triglyceride and HDL cholesterol [5,6,9] was evident (r = -0.54. P < 0.05) during baseline. Drug adherence statistics are also presented for the four treatments in Table 1. No significant differences in adherence were found among the treatments (Fs as = 2.52, P > 0.05). There were also no significant drug sequence effects indicating that the order in which the subjects took the drugs did not affect their lipid or apoprotein responses to the drugs.

4.0 + 44 *

0.72 18

6

+ 50 f 51 f 30

+

N.S.

268 200 135

30 + N.S. 0.76 4.4 f 17 50 * N.S. 89 + 12%

6

i- 75 * 199 ? 27

4.1 * 47 f

32

316 206 129

[191.

0.74 18

5

r 81 336 i- 356 185 + 31 93 P < 0.004 33 + 6 26 P < 0.004 4.2 f 0.74 3.6 47 * 17 26 P < 0.004 92 f 13%

314 268 139

n= 11

Control

n=12

N.S.

285 255 134

mu&z

5 0.47 9

f + *

+ 78 + 206 ? 21

0.67 19

6

+ 85 f 409 k 32

TRIGLYCERIDE,

31 f N.S. 0.55 4.4 + 16 44 f N.S. 81 * 17%

5

Control

* 62 ? 144 f 34

+

4.2 f 45 *

31

t 77 + 172 + 25

Oxandrolone Drug

6

f 0.50 * 12

*

302 209 132

n= 14

Colestipol

Treatment

? 71 275 f 184 124 * 22 137 P < 0.04 c 32 k 7 43 P < 0.004 4.0 f 0.62 3.2 41 * 17 49 P < 0.008 91+ 9%

306 254 126

n= 14

Control

Drug

Control

CHOLESTEROL.

PAS-C

ON PLASMA

Clofibrate

Treatment

AND OXANDROLONE

a AR lipid and protein levels are expressed in mg/dl. b Mean f SD. ’ Two-tailed significance level of difference between drug and control, Fl.88

+

AI/AH HDGCHOL

Adherence

0.72 18

6

r 68b f 190 f 23

32

299 241 123

Baseline

A-II

CHOL TRIG A-I

plasma a

Adherence

4.0 f 44 f

AI/AH HDL-CHOL

?

32

A-11

+ 68b f 190 + 23

299 241 123

CHOL TRIG A-I

Plasma a

Baseline

EFFECTS OF CLOFIBRATE, PAS-C, COLESTIPOL, HDL-CHOLESTEROL AND ADHERENCE

APOLIPOPROTEINS

A-I, A-H,

220

Clofibrate Treatment with clofibrate raised the average concentration of HDL cholesterol above the baseline (predrug) average by 16 f 20% (mean f SD), A-I by 11 + 13%, and A-II by 39 f 17% (Fig. 1). One subject whose HDL cholesterol declined considerably during clofibrate therapy from 93 to 73 was a hyperalphalipoproteinemic woman whose A-I level also declined slightly from 170 to 159. All subjects, however, demonstrated a rise in A-II during treatment. The predominant effect of clofibrate upon A-II was also reflected in the reduced average A-I/A-II ratio (4.00 to 3.20) (Table 1). Clofibrate significantly raised HDL cholesterol 8 mg/dl from its control mean of 41 to 49 mg/dl (Table l), (F i,ss = 12.8, P < 0.008); A-I by 11 mg/dl (F1,ss = 7.29, P < 0.04); and A-II by was no longer correlated 11 mg/dl (&,s~ = 59.9, P < 0.004). Triglyceride inversely with HDL cholesterol during treatment (r = O.lO), nor did the change in triglyceride significantly inversely correlate with the change in HDL cholesterol (r = -0.44). During treatment with clofibrate (as indeed with any of the other 3 drugs), the HDL response of a given subject bore no clear relationship to his or her form of hyperlipoproteinemia, whether classified according to lipoprotein pattern or familial distribution. Oxandrolone This drug exerted uniform and powerful effects upon HDL levels and composition (Fig. 1 and Table 1). HDL cholesterol declined by 36 + 20% from its baseline value, A-I by 21+ 13%, and A-II by 16 f 11% (Fig. 1). Oxandrolone significantly lowered HDL from its control mean of 47 to 26 mg/dl (Table 1) (F i,ss = 80.6, P< 0.004); A-I was lowered by 45 mg/dl (F1,ss= 116.5, P< 0.004); and A-II by 7 mg/dl (F1,ss = 22.2, P < 0.004). The drug rendered subjects with low HDL levels in the baseline state profoundly hypoalphalipoproteinemic (to a minimum of 12 mg/dl of HDL cholesterol in one subject) and

Pretreatment Clofibmte

Pretreatment

I Oxandrolone

Pretreatmeni Clofibrote

1 Oxondrolone

+60-

-6OL

I A-I

A-II

HOL-Chdesterol

Fig. 1. Percentage change of apolipoproteins A-I. A-II and HDL cholesterol of each subject during clofibrate and oxandrolone treatment. Each value represents the mean of 3 samples taken at monthly intervals. Open square represents the mean percentage change.

221

reduced the HDL cholesterol (93 mg/dl) of even the hyperalphalipoproteinemic woman (a longdistance runner) to 47 mg/dl, a level below the female population mean [9]. The A-I/A-II ratio was not substantially changed during oxandrolone. As with clofibrate, triglyceride did not significantly correlate with HDL-cholesterol during oxandrolone (r = -0.41), nor were the changes in each correlated (r = -0.23).

PAS-C Changes in HDL levels and compositions were minimal with this drug. No significant changes in A-I, A-II or HDL cholesterol were evident. The A-I/A-II ratio increased slightly (11 + 15%), while the HDL cholesterol/(A-I + A-II) ratio decreased (6 f 9%). Triglyceride was not significantly correlated with HDL cholesterol during PAS-C (r = -0.40), nor were the changes in these two variables (r = -0.33). Coles tip01 Like PAS-C, this drug exerted minimal effects on HDL levels and composition. HDL cholesterol was increased from baseline values by 6 + 9% and the A-I/A-II ratio was slightly higher than before treatment by 12 ? 12%. Although the correlation coefficient between triglyceride and HDL cholesterol was the same during colestipol treatment as during baseline (r = -0.54), this was not statistically significant because of the smaller number of subjects who underwent colestipol treatment. As with the other drugs, the magnitude of the change in triglyceride during colestipol treatment was not significantly correlated with the change in HDL cholesterol (r = -0.22). Discussion For nearly three decades investigators have sought lipid-related indices of atherosclerotic risk that would be more powerful than the serum cholesterol concentration alone. This search led to the fractionation of lipoproteins into VLDL, LDL, and HDL classes by various techniques. Because of the ominous cardiovascular prognosis of subjects with familial hypercholesterolemia, attention in the past decade focused chiefly upon the increased risk associated with high LDL levels. In the present decade, however, attention has shifted to the HDL, as analysis of retrospective and prospective epidemiological studies in Hawaii [ 41, Framingham [ 51, and elsewhere [ 201 has repeatedly demonstrated the powerful inverse association between HDL cholesterol levels and cardiovascular prognosis. Consequently, current investigations are appropriately concentrating on the effects of various potential modes of intervention, such as diet, exercise, and drugs, upon HDL as well as LDL cholesterol levels, and prospective studies will evaluate whether such changes achieve the changes in cardiovascular prognosis predicted from the population-based studies. At the same time, studies of the interaction between the major apoproteins of HDL, A-I and A-II, have suggested that measurements of those apoproteins may provide important information beyond that offered by HDL cholesterol measurements alone [10,12]. Part of that added information relates to the potentially confounding inverse relationship between HDL cholesterol and

222

plasma triglyceride levels apparent in both healthy persons and in survivors of myocardial infarction [9,10]. Measurements of these apoproteins may also afford additional insight into the differential effects of such interventions upon HDL-hydrated density subclasses. Of the two major subfractions HDLz is relatively enriched in cholesterol and A-I, as reflected in its higher cholesterol/ (A-I + A-II) ratio, and A-I/A-II ratio [12,21]. Furthermore, HDLz is differentially increased in women vs. men, as it is by exogenous estrogen [9] and exercise [22,23]. Hence, though containing a relatively small fraction of total HDL cholesterol, A-I, and A-II, HDL, may contribute disproportionately to the inverse relationship between total HDL cholesterol levels and cardiovascular risk. The widespread prescription of hypolipidemic drugs, often without clear knowledge of their effect on HDL levels and composition, underlined the potential importance of the present study. The studies of the effects of hypolipidemic drugs upon HDL reported to date have been limited to measurements of HDL cholesterol. Nicotinic acid appears to increase HDL cholesterol through a selective rise in HDLz mediated by a reduction in HDL catabolic rate [ 241. Clofibrate has been reported to increase HDL cholesterol in some subjects [25-271, while in others this agent appears to exert little effect [28,29]. Oxandrolone has been shown to reduce HDL in hyperlipidemic subjects [30, 311. Thus studies of the effects of currently prescribed hypolipidemic drugs upon HDL reported to date remain incomplete and the present study substantially advances knowledge in this area. Regarding possible differential effects of these drug on HDLz and HDL3, the greater and more consistent rise in A-II than A-I with clofibrate suggests a selective increase in HDL3. Nevertheless, since the concentration of HDLz is generally considerably lower than that of HDL3, it is possible that clofibrate also increases HDL2. On the other hand, oxandrolone appears likely to reduce both HDL? and HDL3. More subtle differential changes in HDL subfractions may have occurred with the other two drugs. Both PAS-C and colestipol may have increased the HDL2/HDL3 ratio in some subjects, since the mean A-I/A-II ratio was slightly raised in the course of taking these agents. Additional studies of clofibrate and oxandrolone will also be required to elucidate the mechanism responsible for the observed changes in HDL. Both drugs have previously been demonstrated to exert multiple effects on lipoprotein lipase activity [ 32,331 and diminish hepatic triglyceride synthesis [ 341; and oxandrolone to increase post-heparin plasma hepatic triglyceride lipase [ 351 and facilitate triglyceride removal [ 36,371. Hence, such additional studies should address whether these drugs each exert their multiple effects at a single metabolic control point central to VLDL, LDL, and HDL metabolism or at separate points in the metabolism of these lipoprotein classes. Acknowledgements We thank G. Russell Warnick and the Northwest Lipid Research Clinic Core Laboratory for assistance in lipid and lipoprotein analysis. We are indebted to Janice Hoffard, R.N., Maybelle Wagner, R.N., and Ann Grant, R.D., of the Clinical Research Center of Harbor-view Medical Center.

223

William R. Hazzard is an investigator of the Howard Hughes Medical Institute. John J. Albers is an Established Investigator of American Heart Association. Supplementary Description

Material

of Statistical

Analysis

(A) Experimental design This experiment randomly allocated study participants to one of three groups in which the order of drug administration (drug sequence) was balanced. Observations on each subject were made during a baseline period and four treatment periods each consisting of a control and drug interval. This arrangement resulted in a two-factor experimental design. One factor was drug treatment which was of primary interest in the study and the second factor was the drug sequence which was of little interest unless major effects were noted to be due to this factor, Repeated measures occurred on the first factor only; i.e., each subject was observed for each drug treatment but in only one drug sequence. There were a total of 9 measurement periods for 14 subjects or 126 cells in the experimental design. Initially 15 subjects were planned for this study but apolipoprotein measurements were made on only 14. In addition, several participants did not complete their entire drug sequence resulting in smaller sample sizes for colestipol and oxandrolone. (B) Analysis of variance The linear model for this analysis was: Xijk = p + Ai + rk(i) + Bj + ABij + Brjk(i) +

Ek(ij)

where Ai denotes the drug sequence effect, nk(i) denotes the effect of subject k in drug sequence group ai (the experimental error between subjects), Bj denotes the drug treatment effect, ABij denotes the interaction between a drug’s effect and the order in which it was administered, and B7rjk(i) denotes the experimental error within subjects. Since the experimental design included control (drug-free) periods in which the lipids and apolipoproteins returned to baseline values, carry-over effects did not have to be estimated in the model. In this model, “total” variation can be partitioned into two parts. One part experimental error variance plus main consists of the “between subjects” effects due to drug sequence. The second part consists of the “within subjects” experimental error variance plus main effects due to drug treatment and interaction between drug sequence and treatment. Thus, drug sequence effects were tested by comparing the drug sequence mean squared error to the “between subjects” error term. Drug treatment effects and the interaction between drug sequence and treatment were tested effects were using the “within subjects” error term. Once drug treatment established, comparisons between each drug and its control were made using the ratio of their mean differences to the “within subjects” experimental error. Corresponding probability levels were adjusted by a factor of 4 since 4 tests (one for each treatment compared to its control) were performed giving conservative P-values. Homogeneity of variance assumptions were tested and found

224

adequate before proceeding with the analysis of variance calculations original measurement scales for the observations. (C) Analysis of variance results (1) HDL-cholesterol (a) ANOVA table Source of variation

d.f.

F

MS

P

Between subjects Drug sequence Experimental error

13 2 11

3921.9 1824.9

2.15

>O.lO

Within subjects Drug treatment Interaction Experimental error

112 8 16 88

421.8 34.5 33.7

12.5 0.98

CO.01 N.S.

(b) Multiple comparisons, F1 ,ss Estimated

variance for difference MS exp.error

Clofibrate:

I

> 12.8) < 0.002 X 4 = 0.008

(44-45)2 1 == 0.20 33.7[ h + A] 4.999 > 0.20) > 0.50, N.S.

(50 - 47)2 = 2 = 1.67 %.7[i’s + A] 5.4 Pr(F,,,s

Oxandrolone:

n2

64 == 12.8 33*7[T_: + A] 4.999

Pr(F,,,,

Colestipol :

1

two means:

(49 - 41)2

Pr(F,.,s

PAS-C :

1

-+[ nl

between

> 1.67) > 0.50, N.S.

121 (47 - 26)2 == 80.62 33.7[& + A] 5.47 Pr(F,*ss > 80.62) < 0.001 X 4 = 0.004

using the

225

(2) A-I (a) AN0 VA table

Source

of variation

Between subjects Drug sequence Experimental error

Within subjects Drug treatment Interaction Experimental error

d.f.

F

MS

13 2 11

10.105.5 5095.6

P

1.98

>O.lO

112

8 16 88

1981.6 294.7 111.9

11.1 2.6


Significant (P < 0.05) A-I drug sequence-treatment interaction effects called for tests on the simple main effects rather than direct tests on the main effects. Thus differences due to the drug treatments were tested within each of the three drug sequence groups and gave the following results:

al a2

83

MSb at ai

F8.88

P-value

1397.5 981.2 402.0

12.5 a.11 3.59

0.001 0.001 0.006

As can be seen drug treatment differences remain and are even more pronounced (as compared to Fs,ss = 17.7, P < 0.01) when analyzed within each d_rug sequence group. In addition, the Oxandrolone control mean for A-I values (X = 139) is significantly (P < 0.01, Newman-Keuls multiple comparison test) greater than baseline and other treatment control means. This would contribute to the already large difference between the Oxandrolone drug and control means.

(b) Multiple comparisons, F, ,ss Clofibrate:

(137 - 126)2 = E = 7.29 111.9[,: + A] 16.6 Pr(F,,ss

PAS-C :

X 4 = 0.0447 < 0.05

(134-132)’ _ 4 _ o 24 111.9[& + T_:] 16.6 * Pr(F,,ss

Colestipol:

> 7.29) z 0.0112

> 0.24) > 0.50, N.S.

(129--35)2 _ 36 _ 201 111.9[& + A] 17.93 Pr(F,+ss > 2.01) > 0.50, N.S.

226

2116 -(93 139)2 == 116.5 111.9[h + A] 18.17

Oxandrolone:

Pr(F,,ss (3) A-II (a) ANOVA

Source

> 116.5) < 0.001 X 4 = 0.004

table

of variation

MS.

d.f.

F

P

Between subjects Drug sequence Experimental error

13 2 11

684.9 190.0

3.6

Within subjects Drug treatment Interaction Experimental error

112 8 16 88

266.7 12.6 13.6

19.6 0.93

(b)

0.05

< P < 0.10


Multiple comparisons (43 -

Clofibrate :

13.6[A

22)2 + &]

121 == 59.9 2.02

Pr(F,*ss > 59.9) < 0.001 X 4 = 0.004 (3113.6[A

PAS-C:

(30 -

Colestipol:

13.6[A Pr(F,,ss --(26 13.6[h

Oxandrolone:

31)* = 0 N S + A] ’ ’ ’ 32)* + A]

4 = = 1.83 2.18

> 1.83) > 0.50, N.S. 33)* = 49 = 22 2 + $1 2.21 ’

Pr(F,*ss > 22.2) < 0.001 X 4 = 0.004 (4) Adherence

(a) ANOVA table ___df

M.S.

F

P

Between subjects Drug sequence Experimental error

13 2 11

111.6 221.8

0.53

P > 0.10

Within subjecects Drug treatment Interaction Experimental error

42 3 6 33

344.4 116.3 136.8

2.52 0.043

0.06

0.10

Source

of variation

< 0.10

227

HDL cholesterol values

Clofibrate

Baseline

41 50 13 41 70 53 31 53 59 34 33 31 52 44

44 37 93 36 72 4s 26 47 45 25 33 39 44 31

Xzxxi-

-

n

-

Z Change above baseline

Difference

6.82 35.13 -21.51 13.89 -2.18 10.42 42.31 12.71 31.11 36.00 0 -5.13 18.18 41.93

3 13 -20 5 -2 5 11 6 14 9 0 -2 8 13

15.65%; SD =

cx;

- nz’ n-l -_ 14( 15.65)2 13

= = 19.52% Mean and SD was rounded

to be 16% f 20%

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