The impact of homocysteine, B₁₂, and D vitamins levels on functional neurocognitive performance in HIV-positive subjects

Analytical transversal study including fifty-seven (57) caucasian participants with HIV infection who were under continuous ART, followed at the Clinic of the Infectious Diseases, Department of Medicine and Science of Ageing, “G. d’Annunzio” University (Chieti-Pescara, Italy) was performed.

Patients were clinically stable and virologically suppressed and had a CD4+ cell count > 300 cells/mL during the six months before the beginning of the observational period. The patients had no opportunistic infections during this time and no antiretroviral therapy changes in the year before the study started.

After that laboratotory and neurocognitive tests were performed, the patients received a D vitamin supplementation if they showed D hypovitaminosis, in accordance with the current guidelines.

During their baseline visit, blood sample was taken for routine laboratory analysis. Moreover, a clinical assessment was performed. Patients were advised to report any adverse events or changes in their condition throughout the study and to continue their usual diet and lifestyle. Excluding criteria were: (a) steroids, growth hormone, testosterone or any anabolic agent use in the previous six months; (b) drug and alcohol abuse; (c) acute infection in the previous three months; (d) kidney disease and reduced glomerular filtration rate; (e) acute hepatic disease and (f) previous diagnosis of HAND.

This study was conducted in accordance with the guidelines proposed in the Declaration of Helsinki. All participants gave informed consent for the study and the study was approved by the Ethics Committee at the University “G. d’Annunzio” Chieti-Pescara (Ethics Committee Project No.3 the 06/02/2012) and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Overnight fasting venous blood samples were collected for immunological analyses during the intervention period for analysis.

All patients underwent a comprehensive neurocognitive test evaluating memory, attention and working memory, executive functions, speed of psychomotor processing, and language. [14].

The following tests were used to examine the abovementioned neurocognitive skills. For memory: Immediate and Delayed recall of Rey’s words, Delayed recall of Rey’s figure; speed of psychomotor processing was evaluated through Grooved Pegboard Test for both dominant and non dominant hand, attention and working memory through Digit span Forward, Spatial span Forward, Double Barrage, executive functions though Stroop test, Trail Making Test B, Drawings, and WAIS Digit Symbol and language through Letter fluency. The Zung Depression scale in order to assess a mood disorder and the Instrumental Activities of Daily Living (IADL) Scale were also administered.

A trained medical doctor (KF) administered and scored all tests, adjusting them for age, gender, and education on the basis of data available for the general Italian population.

A mild cognitive impairment was diagnosed with a score lower than the normative cut-off in more than three tests [15].

We collected fasting venous blood samples from the antecubital vein of patients at their first examination, determining complete blood count, creatinine, plasma levels of triglycerides, total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, glucose, and albumin. Laboratory tests were performed at our hospital in the Division of Clinical Pathology.

Serum D vitamin concentrations were measured in EDTA by radioimmunoassay, DiaSorin, Balton -UK – according to the manufacturer’s instructions. B₁₂ plasma vitamin and serum folate were quantified using an electrochemiluminescence immunoassay on a cobas e analyzer (Roche, Penzberg, Germany). Total homocysteine was measured in plasma samples by stable isotope dilution liquid chromatography tandem mass spectrometry using a Quattro micro instrument (Waters Corporation, Milford, MA, USA), as described by Magera et al. [16]

Plasma viral load (HIV-RNA) was determined using the “Amplicor” method (Roche Molecular Diagnostics, Milan, Italy) with a sensitivity of 40 HIV RNA copies/mL of plasma. CD4 + − and CD8 + −T cell counts were obtained by flow cytometry of lymphocyte subpopulations.

According to the main endpoint of the study, the sample size determination was based on the prevalence of hyperhomocysteinemia in HIV population based on previously observed data and published results [17]. Assuming a prevalence of 70%, with a precision of 10% and a 95% confidence interval, at least 57 patients were needed. Quantitative variables were summarized as mean and standard deviation (SD) or median and interquartile range (IQR) according to their distribution; qualitative variables were summarized as frequency and percentage. A Shapiro-Wilk’s test was performed to evaluate the departures from normality distribution for each variable. Mann-Whitney U-test was performed to evaluate differences of Homocysteine, B₁₂, and D vitamins between patients with and without pathological value of cognitive test. Logistic regression models were performed to estimate the relationship between Homocysteine, B₁₂, and D vitamins and ANI diagnosis. The results of models were expressed as odds ratio (OR) and relative 95% confidence interval (95% CI). The number of type I errors, was controlled applying the Gavrilov-Benjamini-Sarkar procedure to bound the false discovery rate (FDR) ≤0.05. All statistical tests were evaluated at an alpha level of 0.05. Statistical analysis were performed using IBM® SPSS Statistics v 20.0 software (SPSS Inc., Chicago, Illinois, USA).

A total of 60 Italian participants (50 males, 83.3%), with 51.0 ± 9.8 years of age and 10.8 ± 3.1 years of mean education were enrolled. Three participants were not included into analyses due to incomplete serum sampling. All patients were in cART without therapeutic changes for more than 12 months. All patients were on cART according to currently accepted guidelines. Viral load was persistently undetectable and the median CD4+ cell count was 646 cells/μl (IQR 523–964 cells/μl). The mean serum concentrations of main metabolic values, such as glucidic and lipidic parameters, showed no meaningful discrepancies. Markers of liver and kidney functions did not show any substantial change compared to a range of normality. Furthermore, the mean BMI (Body mass index) was 25.7 ± 3.6 kg/m² (Table 1).

The mean value of homocysteine was 15.1 ± 6.3 μmol/l and high levels of homocysteine (plasma level ≥ 12 μmol/l) [18] were found in 40 participants (70.2% of cases). The mean level of B₁₂ vitamin was 378.5 ± 151.8 ng/ml with levels under 200 ng/L in 8 participants (14.0%); vitamin D mean value was 26.8 ± 9.3 ng/ml. No patients had an alteration in all of the three parameters. The folate levels were normal in all patients, with a mean value of 9.8 ± 13.7 ng/ml.

All cognitive impaired patients showed an ANI profile type, without significant interference in the everyday life (IADL≥7). By analyzing the proportion of impairments, which scored below the normal cut-off, for each single task, median values did not significantly differ with the normal parameter, but 42 patients (73.7%) showed three or more pathologic tasks with ANI: 33 patients had more than 2 pathologic tests (57.9%), 24 patients had more than three pathologic tests (43.6%) and 16 patients had more than 4 pathologic tests (29.1%). The overall tests assessment showed that 21.1% of patients had pathological result in the battery exploring Memory tests and 19.3% of them showed problems in long time-memory; 66.7% of patients showed a pathological test of attention and working memory, 57.9% was pathologic on executive functions, 31.6% had low points on speed of psychomotor processing. For 12 patients (21.1%) there was a significant pathologic result in Zung Depression scores. Raw scores for each task and comparison cut-off are illustrated in Table 2.

Abnormal homocysteine levels were associated with worsened performance in verbal fluency (p = 0.003) and worse executive functions (Stroop E test p = 0.040). 25-OH D hypovitaminosis was associated with worse performances in executive functions in three different test: Stroop E (p = 0.049), Trail B (p = 0.035) and Wais Digit Span (p = 0.042). Pathological levels of B₁₂ vitamin were also associated with worse performances in executive functions (Trail B Test and Wais Digit Span respectively p = 0.002 and 0.029) and with lower speed of psychomotor processing (Peg Board Test on dominant hand, p = 0.014) (Table 3). No significant relations were found among pharmacological therapy, HBV/HCV co-infections and vitamins levels. The group that received an ANI diagnosis (over 3 pathologic neurocognitive tests) showed lower levels of 25-OH D vitamin compared with the patients that had less than 3 pathologic tests (p = 0.022) (Fig. 1). No difference in homocysteine levels and B₁₂ vitamin levels were found between patients with less and more than 3 pathological cognitive tests.

Logistic regression analysis confirmed the protective activity of 25-OH D hypovitaminosis on the risk of ANI (OR = 0.891; p = 0.042), as showed in Table 4.