Effect of DHA on Lipid and Carbohydrate Metabolism Alterations and Body Fat Distribution in HIV Patients Under HAART.
Effect of DHA on Lipid and Carbohydrate Metabolism Alterations and Body Fat Distribution in HIV Patients Under HAART.
Highly active antiretroviral therapy (HAART) is able to cause lipid metabolism and glucose
 homeostasis alterations, which are associated to the redistribution of body fat. Alterations
 in lipid and carbohydrate metabolism contribute to the development of a highly atherogenic
 profile, which together with altered fibrinolysis markers and increased presence of
 proinflammatory cytokines in blood (especially tumor necrosis factor alpha) that comes
 associated to the success of HAART can cause the development of accelerated atherosclerosis.
 Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that has demonstrated its ability
 to reduce triglyceride levels; modify cholesterol fractions and increase the size of LDL
 particles thereby configuring less atherogenic plasma profile. Additionally, administration
 of DHA has shown antiinflammatory and hypotensive activity, which contributes to reduce the
 risk of cardiovascular complications in these patients. At a molecular level, DHA acts as a
 stimulator of the nuclear receptor PPAR-gamma, which has been described to induce an increase
 in adipocyte differentiation. Furthermore, the anti-inflammatory effects induced by DHA, can
 decrease the elevated levels of TNF-alpha, which has been implicated in the pathogenesis of
 body fat redistribution in HIV infected patients undergoing HAART. Therefore, the hypothesis
 of this project is that DHA will be able to produce lipid-lowering, anti-inflammatory,
 hypotensive and profibrinolytic effects, which all together should improve atherogenic
 profile of patients with HIV-1 infection receiving HAART. In addition, their proprieties as
 PPAR agonist can improve the redistribution of body fat present in many of these patients.
 The study of the activity of DHA on dendritic cells and monocytes should indicate the absence
 of immunosuppressive effect of DHA in the context of HIV-1 infection.

 In summary, DHA is a natural product, from the omega 3 polyunsaturated fatty acids, the
 therapeutic properties of which have been described in recent years and has shown
 cardio-vascular and metabolic beneficial effects, without recognized side effects. The highly
 purified DHA administration at high doses could be able to reverse, at least partially, lipid
 abnormalities associated with HAART and to exert a beneficial effect on fat redistribution in
 HIV-infected patients treated with HAART. To ensure non deleterious immunological treat in
 these sensitive poly-medicated patients, substantial changes in the functionality of
 dendritic cells and monocytic will be studied.
Hypothesis: DHA treatment would be able to revert, at least partially, the lipid disturbances
 associated with HAART and to improve or at least not to worse fat redistribution associated
 with HAART, without inducing further derangements to dendritic cells and monocyte functional
 ability.

 Variables: effects of DHA treatment on total cholesterol and its fractions, triglycerides,
 insulin, fibrinolysis markers, tumor necrosis factor alpha (TNF-α), and fat distribution
 assessed by anthropometric measurements, bioimpedance, sonography, DEXA and abdominal CT.
 Effects of DHA administration on phenotype and functional capabilities of dendritic and
 mononuclear cells. Objective: To determine if treatment with high doses of highly purified
 docosahexanoic acid (DHA) is able to revert, totally or partly, the lipid disturbances and
 the fat redistribution associated with highly active antiretroviral therapy (HAART), and the
 effects on phenotype and function of dendritic cells and monocytes Data analysis: Baseline
 data will be analyzed for a good balance. Fisher´s exact test will be used to assess
 differences between categorical variables, Student's t test for continuous variables, and
 Mann-Whitney test for ordinal variables. The main efficacy variable will be differences
 between baseline and final values using MANCOVA model taking baseline value as covariable.
 Efficacy will be assessed by contrasting adjusted means at the end of the study between both
 groups of treatment. Intention to treat analysis will be used.

 The methodology to perform in the centers where the clinical trail will be conducted are the
 following: After obtaining informed consent, the patient's baseline will be examined with the
 collection of demographic data, HIV infection status, pharmacological data, anthropometric
 parameters, and it will proceed to conduct a complementary examination designed to identify
 and define body fat composition, body fat distribution and to perform a basal analysis
 including the parameters previously mentioned. It also will be evaluated the
 hygienic-dietetic habits of the patient with special attention to alcohol intake, physical
 activity and concomitant medication. This procedure will be carried out by making
 retrospective diaries from the last 7 days. The patient's daily caloric intake will be
 quantified by the Nutrilogic ® software (Bio Logic, Barcelona, España). Physical activity
 will be quantified by the Minnesota scale (Elosua R, Marrugat J, Molina L, Pons S, The
 MARATHOM Investigators. Validation of the Minnesota leisure time physical activity
 questionnaire in Spanish men. Am J Epidemiol 1994, 139: 1197-1209).

 Body fat composition and body fat distribution: will be done with the following additional
 tests:

 Densitometry or DEXA: Will be determined with the patient in supine position with legs
 straight and feet together, on a standardized examination table from a densitometry device
 (Lunar Prodigy, Madison, WI, USA). In the initial study enerarán more faces of the different
 regions that will be copied and transferred to the images of subsequent studies to reduce the
 variability of successive determinations. There will be performed a body fat measurement,
 body composition and assessment of bone mineral density by dual absorptiometry technique.
 There will be performed a total body "scan" (body composition) or focused on lumbar spine
 (trabecular bone) and proximal third of the femur (cortical bone) for the evaluation in the
 last two cases of bone mineral density and a possible osteopenia or osteoporosis.

 Abdominal CT: Will be obtained by performing a spinal digital radiography, latero-lateral
 view, locating L4 and getting a tomographic section passing through the center of the
 vertebral body. Subsequently, there will be a digital calculation of intra-abdominal fat and
 subcutaneous fat, manually selecting the area of interest. The areas measured will be done in
 cm2.

 Lab analysis: blood samples for biochemical determinations will be obtained in Vacutainer
 tubes of 10 ml without additives. Blood will be clotted at room temperature for 30 minutes
 and centrifuged at 2500 rpm for 15 minutes. After separating the serum from the clot (about
 4-5 ml), will be divided into 1 aliquot of 1.5 ml (for the study of lipids) and 5-7 aliquots
 of 0.5 ml aliquots which will be frozen at -20 °C until the biochemical analysis are carried
 out.

 Plasma lipids, glucose, glycosylated hemoglobin, insulin and C-peptide: measurements of total
 cholesterol and triglycerides will be performed using automated trading methods (enzyme
 colorimetry, Roche Diagnostics) adapted to the automated autoanalyzer Hitachi 911. The
 determination of apolipoproteins AI, B and C-III, lipoprotein (a) will be held mediate
 commercial methods (immunoturbidimetric assay, Roche Diagnostics and Wako Chemicals) adapted
 to the automated autoanalyzer Hitachi 911. The determination of cholesterol lipoprotein
 fractions (Lipid, ELIP) (VLDL, LDL, HDL) will be performed by the following methods: VLDL
 separated by ultracentrifugation of flotation, cholesterol HDL determined by a direct method
 (Roche Diagnostics) and cholesterol LDL determined by difference in total cholesterol and
 VLDL and HDL cholesterol fractions. Glucose: by a commercial method (enzyme colorimetry,
 Roche Diagnostics) adapted to the automated autoanalyzer Hitachi 747. Serum insulin
 concentration will be determined by non-competitive chemiluminescent enzyme immunoassay
 (Immulite 2000TM, Diagnostic Products Corp., Los Angeles, CA, USA). The serum concentration
 of C-peptide is determined by non-competitive chemiluminescent enzyme immunoassay (Immulite
 2000TM, Diagnostic Products Corp., Los Angeles, CA, USA).

 Determining the size of LDL particles: The size of LDL particles will be determined from
 total plasma using acrylamide gradient gel electrophoresis under nondenaturing conditions.
 Will be used a standard of four known LDL diameter bands, previously determined by electron
 microscopy.

 Determination of fatty acids in plasma: Treatment compliance will be corroborated by
 determining the fatty acid composition of plasma lipids by gas chromatography. Plasma total
 lipids will be extracted by the method of Bligh and Dyer with chloroform: methanol (2:1, V:
 V). Methyl esters of fatty acids will be obtained by transesterification with boron
 trifluoride in methanol at 80 °C for 60 min. which allows a high recovery of all the lipidic
 compounds including PUFAs. Aliquot containing the methyl esters of fatty acids will be
 analyzed with a gas chromatography device (Hewlett-Packard model 5890) on a 30 m. RTX-2330
 column (Restek, Bellefonte, PA) with an internal diameter of 0.25 mm equipped with a flame
 ionization detector. The carrier gas will be helium at a pressure of 105 kPa. To the total
 separation of the different compounds it has provided with two temperature settings: 140
 °C-200 °C to 3 °C/min or in two stages of 140°C-180°C to 4°C/min and 180°C-210°C at 2°C/min.
 The temperature of the injector and detector is 260 °C. The linear response of the detector
 will be tested periodically with standard mixtures. Typically, two internal standards with
 different molecular weight (13:0 and 23:0 or 27:0) will be used. Peaks will be integrated
 with an integrator D-2500 (Hitachi Ltd., Tokyo) and identified by comparison of retention
 times with standards. When necessary, the identification may be confirmed by mass
 spectrometry (Hewlett-Packard detector model 5970B) at an ionization potential of 70 eV.

 Study of markers of inflammation: blood will be withdrawn in 5 ml EDTA tube. After
 extraction, the tube will be centrifuged, and plasma collected in eppendorf tubes and frozen
 at -70°C until cytokines determination. The content of TNF-alpha, IL-1 beta and IL-6 in the
 plasma samples will be measured by ELISA. Each sample will be analyzed in duplicate using
 ELISA kits "R & D Systems" following the specifications that commercial supplier.

 Intervention: Patients will be randomized to one of two treatment groups (placebo and DHA),
 using a centralized randomization list. The study will be conducted according to the
 double-blind trial methodology, in which neither the patient nor the physician are unaware of
 the active substance administered to the patients. The randomization procedure will be
 performed assigning either strategy according to a randomization scheme generated by the
 module PROC PLAN SAS (version 8.2) in multiple of 2 blocks and following a pattern 1:1
 stratified by center. A list of random assignment will generated according to the above
 mentioned, and as from the same will be issued a sealed opaque envelope, identified by a
 sequential number and the name of the center. The envelopes shall be deposited in the center
 of central data management and analysis, which will act as coordinator of the centralized
 randomization assignment by telephone contact.

 Patients will be evaluated for inclusion in the study, and their personal data collected in a
 specific collection data notebook. After the informed consent procedure and if the patient
 consents to be included in the study, the centralized randomization assignment center will be
 contacted. When patient data are registered, the envelope will be opened and the strategy
 assigned to the patient reported to the investigator. The procedure shall be transcribed into
 a document contained in the envelope, signing and dating this register the person responsible
 at the central randomization center.

 Capsules with identical external appearance containing oleic acid (placebo) and
 docosahexaenoic acid at doses of 500 mg per capsule will be previously prepared. The capsules
 are coated with a protective cover which makes it taste imperceptible to the patient. The
 dosage of DHA will be of 4 g daily. At the same intervals, patients in the placebo group will
 take 4 g daily of oleic acid.

 Data collection: The data of patients will be collected by a physician devoted entirely to
 guide patients, coordinating tests to perform, and recording information into a database.
 Data will be collected in a previously designed specific collection data notebook.

 o Sample size assessment to specify the number of participants or participant years necessary
 to demonstrate an effect.

 Estimating sample size and data analysis:

 It is expected to be able to show a difference in favor of the active treatment in the main
 variable, namely, reduction in triglycerides values at the end of the follow-up period (48
 weeks) of at least 20% in magnitude, in the comparison of the means adjusted by a covariance
 analysis. According to the estimated standard deviation of 86 from our own data, consistent
 with literature data (AIDS 1999, 13:1424-1425), and the magnitude of the minimum effect
 pre-defined as relevant (20% reduction), it would be enough to some 29 evaluable patients per
 group. It is intended to recruit more patients as forecast of possible expected losses of
 about 10%, or in other words, 33 subjects per group.

 Re-estimation of the sample size:

 Since variability is estimated from data from other situations (pre-treatment baseline data),
 and there is no concrete data on the experimental situation (variability after DHA
 treatment), it is predefined in this protocol to perform a re-estimation of the sample size
 when will be obtained a follow up of the 50% after 4 weeks of treatment of the triglycerides
 values. To this objective data from the main variable, triglyceride levels at baseline and
 after 4 weeks post-treatment, along with the random assignment but masking the codes will be
 provided to a second independent statistical group (Catalan Institute of Oncology, Department
 of Epidemiology and Cancer Registration. Statistician: Dr. Victor Moreno) respect to central
 statistical analysis of this study (Laboratory of Biostatistics and Epidemiology (LBE),
 Section Clinical Trials, Autonomous University of Barcelona. Statistician: Dr. Ferran
 Torres). This new group will perform the analysis, and will only reveal variability data to
 recalculate the sample size. If the sample size were less than expected, it will be
 maintained according to the initial design. Under no circumstances significance tests will be
 performed or the study will be prematurely stopped secondarily to re-estimate variability.
 According to this method, you do not need any adjustment of multiplicity.

 o Plan for missing data to address situations where variables are reported as missing,
 unavailable, "non-reported," uninterpretable, or considered missing because of data
 inconsistency or out-of-range results Treatment of missing values

 The main analysis by intention to treat will require using all subjects without relevant
 deviations from the selection criteria, whom they have taken at least one dose of the studied
 medication and who have evaluation of the main variable prospectively, at least at three
 months of the follow up. Under this assumption, it is expected that in a 1-year study follow
 up find subjects with measurements not made. Therefore it is set out the imputation of
 missing values of the main variable as follows:

 -  If the lack of data is due to problems of efficiency, or the cause is unknown, shall be
 attributed the 95% percentile value for the study group.

 -  In case of missing data for other reasons, will be implemented the LOCF (Last
 Observation Carried Forward) method provided the availability of a value of at least
 three months after its baseline measurement.

 The detailed procedure will be included in the statistical report and it is planed evaluate
 the consistency of the method before closing the database at a meeting at which anonymous
 listings will be facilitated for its evaluation 'Blind Review' where the procedure may be
 re-evaluated (CPMP/EWP/1776/99: Points to Consider on Missing Data).

 Difficulties and limitations of the study:

 The number of patients and the peculiar management that they will have, due to the additional
 tests performed, make the study inevitably complex. However, there will be a physician who
 permanently coordinates all patients and all tests performed. In addition, the research team
 is wide with concrete and synergistic functions for each researcher. Finally, researchers
 constitute a consolidated group and are experienced in previous studies with similar
 requirements.

 o Statistical analysis plan describing the analytical principles and statistical techniques
 to be employed in order to address the primary and secondary objectives, as specified in the
 study protocol or plan.

 Statistical analysis Descriptive analysis

 The following descriptive indexes will be detailed in the statistical report according with
 the nature of the variables:

 -  In continuous variables: Mean, CI 95%, SD, minimum, P25, median, P75, max, N and
 missing. Per group and globally.

 -  In categorical variables: % total with respect to the column, N in each category. Per
 group and globally.

 -  In ordinal variables: They will be described with two tables: one with descriptive
 parameters of the continuous variables and the other with categorical variables.

 In open fields or when are interested to list all cases for one or more variables, will be
 described by lists.

 Inferential analysis The statistical treatment of the primary and secondary efficacy
 variables will be specifically described in a section below. For all other variables, the
 appropriate hypothesis test will be applied according to its nature: Fisher exact test for
 categorical variables, the Student t test for continuous variables, Mann-Whitney U test for
 ordinal variables.

 Basal homogeneity between groups An analysis of the baseline comparability of treatment
 groups will be established regarding demographic variables, risk factors and clinical
 characteristics at the time of inclusion.

 Main efficacy analysis The main variable of efficacy will be evaluated by comparing the
 difference between baseline and end values of the different treatments using ANCOVA model
 with the baseline value of the test as covariate. Adjusted means were calculated and
 confidence intervals at 95% (95%) by the ANCOVA model. As the main decision criterion of
 efficacy the contrast between the adjusted means at the endpoint of the study between the two
 treatment groups will be performed. Additionally and as an exploratory intent, shall be
 evaluated the compared effect at the different times of observation. No multiplicity
 adjustments will be made due to the exploratory nature of this second analysis.

 In case of relevant deviations in the cases pursuant of the parametric tests, it will be
 applied a nonparametric approach using transformations to ranges of the corresponding
 variables. The main analysis of the efficacy will be done with the population intended to
 treat. However, as evidence of sensitivity and robustness of the results, the analysis under
 the protocol approach will be provided.

 Tolerability and acceptability tests All patients who have been randomized and treated will
 be included in the safety analysis. The frequency of appearance of adverse events in the two
 groups will be compared using the Fisher's exact test.

 Level of significance In all proposed statistical tests the level of significance required
 shall be the conventional (p < 0.05 bilateral). No provisions have been made to perform any
 multiplicity adjust except in the intermediate analysis.
Inclusion Criteria:

 1. Serologic evidence of HIV-1.

 2. Plasma triglycerides > 2.26 mmol/L (200 mg/dL) and/or Total Cholesterol > 6 mmol/L
 (232 mg/dL) measured after a fasting period of at least 12 hours and confirmed by at
 least two measures separated by at least an interval of one week.

 3. Body mass index between 25 and 30 kg/m2.

 4. Absence NSAIDs, antihypertensive drugs, lipid-lowering drugs treatments or any other
 medication known to affect plasma lipid levels.

 5. Alcohol intake <20 g/day.

 6. Absence of Diabetes mellitus and obesity (BMI > 30)

 7. Absence of heart and liver pathologies (within 3 months prior the study).

 8. Absence of renal failure (serum creatinine > 130 mmol/L) or Cr.Cl. < 60 ml/min /1.73
 m2.

 Exclusion Criteria:

 1. Pregnancy or absence of adequate contraception in childbearing age women.

 2. Presence or fail to fulfil one or more of the conditions listed in paragraphs 2-8 of
 the inclusion criteria