Decreased Docosahexaenoic Acid Levels in Serum of HIV Carrier Patients

Abstract

The study aimed to measure serum fatty acids (FAs) composition in HIV carrier patients and compare it with non-HIV carrier patients. The FAs composition was measured by gas chromatography as follows: four saturated FAs myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), and docosanoic acid (22:0); four monounsaturated FAs 7-hexadecenoic acid (16:1 n-9), palmitoleic acid (16:1 n-7), oleic acid (18:1 n-9), and vaccenic acid (18:1 n-7); and three polyunsaturated FAs linoleic acid (18:2 n-6), dihomo-γ-linolenic acid (20:3 n-6), and docosahexaenoic acid (DHA, 22:6 n-3). We reported herein lower (P < .05) DHA concentration (by 40%) in the serum of HIV carrier patients than in non-HIV carrier patients. This FA has a pivotal role as a precursor of anti-inflammatory molecules with beneficial effects on metabolism, cardiovascular system, and immunological system. Even though most clinical studies reported beneficial effects of DHA supplementation in HIV carrier patients, this issue remains under debate. Further investigations then require to fully clarify the role of DHA in preventing or alleviating the comorbidities associated with HIV infection.

The World Health Organization's top 10 most common health issues include HIV infection,¹ since the 1980s.² The mortality caused by HIV has decreased since the introduction of highly active antiretroviral therapy (HAART).³ There is no cure for AIDS yet. Thus, strategies to increase the efficacy of the treatment-associated complications of the disease and improve the life quality of HIV carrier patients are then required.^4
–6

Increased inflammation and a higher risk of cardiovascular complications in HIV carrier patients have been reported.^7
–9 This condition is comparable with that of obese non-HIV carrier patients wherein chronic inflammatory and cardiovascular disease is associated with increased blood levels of proinflammatory fatty acids (FAs) and decreased anti-inflammatory FAs.¹⁰

The docosahexaenoic acid (DHA) belongs to the ω 3 (n-3) polyunsaturated FAs (PUFA) family and their nutraceutical properties have been described over the years. Many studies reported their effects on blood pressure, metabolism, immunomodulation, inflammatory process, cancer cachexia, mental illness, and brain development.^10

–13

There are only a few studies^14
–16 measuring serum FAs composition, that is, saturated FAs (SFA); monounsaturated FAs (MUFA), and PUFA in HIV carrier patients. Herein, we compared the serum FAs composition in HIV carrier patients with that in non-HIV carrier patients.

The Maringá State University Ethics Committee (COPEP 1.166.674) and the Brazilian Clinical Trials Register (RBR843tnq) approved the protocol of the study. Inclusion criteria were HIV infection diagnosis, CD4⁺ T cells count <500 cells/mm³, and stable and unchanged HAART for at least 1 year. Exclusion criteria were pregnant women and patients with nephropathies and/or hepatopathies.

All patients signed the informed consent form that includes this information: “The collected blood will be stored in the form of serum or plasma. If there is a need for additional dosages in the serum or plasma, these will be performed as long as they do not interfere with your privacy. We also remember that the information obtained will be used only for research purposes and will be treated with confidentiality to preserve your privacy.” Therefore, serum from all HIV carrier patients (n = 11) from our previous study¹⁷ stored and frozen at −80°C in our laboratory was used. Two samples were excluded because the patients were hyperlipidemic. Details about inclusion and exclusion criteria, biochemical, toxicological, hematological, and inflammatory parameters may be found in our previous clinical study.¹⁷

For comparative purposes, serum samples from 9 normolipidemic non-HIV carrier individuals from the serum bank of immunogenetics laboratory of our university were selected based on these criteria: age and a similar proportion of man/woman in comparison with HIV carrier patients. The main information about HIV carrier patients and non-HIV carrier patients are given in Table 1.

Table 1.

Gender and Age of HIV Carrier Patients (HIV Group) and Non-HIV Carrier Patients (Non-HIV Group)

Patient no.	Gender of HIV group	Gender of non-HIV group	Age of HIV group (years)	Age of non-HIV group (years)	Civil status HIV group	Education level HIV group	Physical activity HIV group	Smoking habit HIV group	Alcoholic behavior HIV group	Diagnosis of HIV (years)	Start current HAART (years)	Current HAART
1	Male	Male	34	31	Married	High school	No	No	No	3	3	3TC+TDF+LPV/r
2	Female	Female	53	51	Divorced	Middle school	Yes	Yes	No	5	1	3TC+TDF+EFZ
3	Female	Female	55	54	Single	Middle school	No	No	No	7	7	3TC+AZT+ATV+RTV
4	Male	Male	45	42	Married	Middle school	Yes	No	No	2	2	3TC+AZT+LPV/r
5	Male	Male	23	25	Single	High school	Yes	No	No	2	2	3TC+TDF+ATV+RTV
6	Male	Male	43	50	Married	Middle school	No	No	Yes	5	2	3TC+TDF+EFZ
7	Male	Male	23	29	Single	Middle school	No	No	No	3	1	3TC+TDF+EFZ
8	Female	Female	40	43	Widower	Middle school	Yes	No	No	17	2	3TC+TDF+EFZ
9	Female	Female	47	48	Married	Middle school	No	No	No	13	2	3TC+TDF+ATV/r

Civil status, educational level, physical activity, smoking habits, alcoholic, diagnosis time, years after starting HAAT, and current HAART.

3TC, lamivudine; ATV, atazanavir; ATV/r, atazanavir/ritonavir; AZT, zidovudine; EFZ: efavirenz; HAART, highly active antiretroviral therapy; LPV/r, loponavir/ritonavir; RTV, ritonavir; TDF, tenofovir.

We employed the method described by Figueiredo et al. ¹⁸ to directly transesterify the FAs into FAs methyl esters (FAMEs) by acid and alkaline catalyzation. The FAME was separated, identified, and measured by gas chromatography following the experimental conditions exposed in our previous publication.¹⁹ Retention times and peak areas were determined using the Chrom-Quest™ software (Thermo Scientific™). The unsaturation index of FAs composition was calculated as previously described by Moreira et al.²⁰

The Student's t-test compared the results expressed as the mean ± standard error of three analyses of FAs. All premises for t-test use were met: normal distribution by Shapiro–Wilk test and the similarity between data dispersion by Fisher's statistics. P values ≤.05 were employed to indicate statistically significant differences between means.

We measured 11 FAs: 4 SFAs, myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), and docosanoic acid (22:0); 4 MUFAs, 7-hexadecenoic acid (16:1 n-9), palmitoleic acid (16:1 n-7), oleic acid (18:1 n-9), and vaccenic acid (18:1 n-7); and three PUFAs, linoleic acid (18:2 n-6), dihomo-γ-linolenic acid (20:3 n-6), and DHA (22:6 n-3) (Table 2).

Table 2.

Concentrations of Serum Fatty Acids and Unsaturation Index in HIV Carrier Patients (HIV; n = 9) and Non-HIV Carrier Patients (Non-HIV; n = 9)

Fatty acids	Groups	Concentration (mg/g ± SE)	P	Δ (%)
Myristic acid (14:0)	Non-HIV	0.032 ± 0.006	.33	−25.0
Myristic acid (14:0)	HIV	0.024 ± 0.006	.33	−25.0
Palmitic acid (16:0)	Non-HIV	0.501 ± 0.050	.69	−6.0
Palmitic acid (16:0)	HIV	0.471 ± 0.051	.69	−6.0
Stearic acid (18:0)	Non-HIV	0.167 ± 0.012	.90	−2.4
Stearic acid (18:0)	HIV	0.163 ± 0.017	.90	−2.4
Docosanoic acid (22:0)	Non-HIV	0.139 ± 0.016	.31	−15.8
Docosanoic acid (22:0)	HIV	0.117 ± 0.012	.31	−15.8
7-Hexadecenoic acid (16:1 n-9)	Non-HIV	0.014 ± 0.003	.18	−28.6
7-Hexadecenoic acid (16:1 n-9)	HIV	0.010 ± 0.001	.18	−28.6
Palmitoleic acid (16:1 n-7)	Non-HIV	0.041 ± 0.006	.77	9.8
Palmitoleic acid (16:1 n-7)	HIV	0.045 ± 0.009	.77	9.8
Oleic acid (18:1 n-9)	Non-HIV	0.386 ± 0.040	.50	13.7
Oleic acid (18:1 n-9)	HIV	0.439 ± 0.064	.50	13.7
Vaccenic acid (18:1 n-7)	Non-HIV	0.029 ± 0.004	.36	20.7
Vaccenic acid (18:1 n-7)	HIV	0.035 ± 0.006	.36	20.7
Linoleic acid (18:2 n-6)	Non-HIV	0.625 ± 0.038	.18	−13.3
Linoleic acid (18:2 n-6)	HIV	0.542 ± 0.044	.18	−13.3
Dihomo- γ-linolenic acid (20:3 n-6)	Non-HIV	0.034 ± 0.004	.72	−5.9
Dihomo- γ-linolenic acid (20:3 n-6)	HIV	0.032 ± 0.004	.72	−5.9
Docosahexaenoic acid (22:6 n-3)	Non-HIV	0.023 ± 0.002	<.01	−39.1
Docosahexaenoic acid (22:6 n-3)	HIV	0.014 ± 0.001^*	<.01	−39.1
SUM	Non-HIV	1.936 ± 0.166	.87	−2.1
SUM	HIV	1.896 ± 0.179	.87	−2.1
Unsaturation Index	Non-HIV	100.40 ± 8.88	.63	−5.8
Unsaturation Index	HIV	94.54 ± 7.900	.63	−5.8

The values are expressed as mean ± SE of three analyses quantified by gas chromatography. Unsaturation index was calculated as described by Moreira et al. ²⁰

P value <.05.

Δ (%), percentage of variation; n-9, ω 9; n-7, ω 7; n-6, ω 6; n-3, ω 3; SE, standard error; SUM, amount of total fatty acids calculated through the sum of each fatty acid concentration.

The main limitation of this study was the small sample size due to the difficulties for the regimentation of HIV carrier patients. Additional limitations of the study include lack of generalizability, impossibility to differentiate the influence of HIV infection, and antiretroviral therapy on serum levels of FAs. Also, the study lacks information about n-3 FAs ingestion and absence of values of plasma levels of others n-3 PUFAs: eicosapentaenoic acid (20:5 n-3) and α-linolenic acid (18:3 n-3).

The very similar (by 2% difference only) sum of FAs (HIV vs. non-HIV carrier patients) allows comparing specific FAs without the jeopardizing influence of differences in the total amount of FAs (Table 2). The serum FAs compositions were not different between groups, except for the DHA concentration. We observed decreased (P < .05) DHA levels (by 40%) in HIV carrier patients (Table 2). To better clarify this result, a figure displaying individual results for DHA was included (Fig. 1).

FIG. 1.

Graph showing individual DHA serum concentrations of non-HIV carrier patients (non-HIV) and HIV carrier patients (HIV). *P < .01 (HIV vs. non-HIV). DHA, docosahexaenoic acid.

DHA is an n-3 PUFA with six double bonds highly susceptible to lipid peroxidation by reactive oxygen species (ROS).²¹ The production of ROS is enhanced in HIV carrier patients.²² The unsaturation index of FAs composition reflects the extension of lipid peroxidation due to consequent reduction in the content of PUFAs.^20,23 Despite the marked decrease in DHA levels, the unsaturation index decreased only slightly (Table 2).

DHA deficiency is associated with inflammation,²⁴ cardiovascular diseases,²⁵ and cancer cachexia.¹² It was suggested that n-3 PUFA within the human diet is pivotal to brain development.²⁶

The HIV infection promotes inflammation wherein decreased serum DHA could be a consequence of chronic inflammation.⁷ We have previously demonstrated that these patients show high C-reactive protein blood levels, a biomarker of proinflammatory state.¹⁷ Nevertheless, one way to prevent unexpected damage associated with a higher proinflammatory state is through anti-inflammatory molecules²⁴ wherein DHA plays an important role.²⁷ Thus, depletion of DHA in HIV carrier patients (∼40%) may be a consequence of an increased demand for anti-inflammatory molecules to alleviate the proinflammatory state promoted by the infection.

The beneficial effects of n-3 PUFA supplementations (mainly DHA as fish oil) on health are well established. For instance, many studies have reported anti-inflammatory effects,^24,27 improvement of immunological response,^28,29 and decrease of the risk of cardiovascular complications.³⁰ In agreement with these clinical trials, decreased interleukin 6, tumor necrosis factor-α, triglycerides, and increased leukotriene B5 serum levels after n-3 PUFA supplementation were reported in HIV carrier patients.^31
–33

In contrast, there is a lack of favorable reports on the effects of DHA supplementation on oxidative stress,³⁴ depressive symptoms,³⁵ and endothelial function³⁶ in HIV carrier patients.

Even though most clinical studies reported beneficial effects of DHA supplementation in HIV carrier patients, this issue remains under debate. Further investigations then require to fully clarify the role of DHA in preventing or alleviating the comorbidities associated with HIV infection.

Footnotes

Acknowledgments

We are grateful to Cristiano Rodrigues Schamber, José Ricardo Colleti Dias, and Tuane Krupek for their contributions.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This research was supported by the Program Research for SUS (PPSUS)/Brazil (Protocol Nos. 41645.433.30361.12092013 and 48083.501.44971.1204.2017). We are also thankful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the Social Demand Grant (No. 88882.448894/2019-01), CNPq, and FAPESP.

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