In 2016, patients of both genders aged from 9 to 61 years, diagnosed with common variable immunodeficiency, according to the criteria of the European Immunodeficiency Society [14], were evaluated through a controlled cross-sectional study, conducted in the Discipline of Allergy, Clinical Immunology and Pediatric Rheumatology, Federal University of São Paulo – São Paulo Medicine School (UNIFESP-EPM).
Thirty-two patients with CVID and 37 healthy volunteers were included, paired with patients by gender and age, comparing nutritional status variables, biochemical markers related to cardiovascular risk (CVR), and food intake.
The study followed the guidelines of the Helsinki Declaration of 1975 as revised in 1996 regarding the use of human subjects. The study was approved by The Research Ethics Committee from the Federal University of São Paulo (No. 972812/2015), with funding received by The São Paulo Research Foundation - FAPESP nº 2015/13308-9, and signed informed consents were obtained from all participants (or a responsible guardian in the case of children).
Clinical, demographic, and socioeconomic data were collected through a structured and standardized questionnaire. Anthropometric and food consumption assessments and biochemical tests were also performed. At the time of collecting laboratory tests, none of the subjects had an acute infectious disease, clinically identified through the complete blood count, nor had they been using corticosteroids for at least three months.
Anthropometric and food consumption assessment
The anthropometric evaluation included measurements of weight, height, skinfolds (bicipital, tricipital, subscapular, and suprailiac, and abdominal circumference. Weight and height were measured according to the recommendations of the World Health Organization (WHO) [15] and skinfolds, according to Frisancho (1990) [16].
The body mass index-for-age (BMI/A) and height-for-age (H/A) indicators were calculated for the classification of nutritional status, expressed as Z-score, using De Onis (2007) [17] as a reference, for children and adolescents, and the body mass index (BMI), as proposed by the World Health Organization for adults (WHO, 1995). The classification by Freedman et al. [18] was used to assess the waist circumference of children and adolescents, and values above the 90th percentile were considered high. Adults were classified according to the WHO [15]. Body composition was estimated based on the sum of the four skinfolds for adults [19] and the TSF and SSSF for children/adolescents using Slaughter’s Eq. (1988) [20]. Body fat percentage was classified as proposed by Deurenberg (1990) [21] and Lohman (1992) [22].
The stage of pubertal development was self-assessed (Saito, 1984) [25], according to Marshall & Tanner (1969) [26].
Food consumption was assessed with the 24-hour food record [27], applied in three stages [28], with a 15-day interval. The surveys were collected on Tuesdays, Thursdays, and Fridays, thus avoiding days after the weekends.
The participants’ food intake was calculated with the Dietwin software, comparing cases to controls. Considering that the food composition tables available in some software do not have complete data about the Se content in foods, these data were included manually based on the paper by Ferreira et al. (2002) [29]. Also, the total consumption of energy, lipids, protein per kilogram of body weight, vitamins A and C, copper, and zinc were analyzed.
Biochemical assessment
After a 12-hour fast, a blood aliquot was collected to analyze selenium, glutathione peroxidase, lipid profile, apolipoproteins A-1 and B, oxidized LDL (LDLox), malondialdehyde (MDA), ultra-sensitive C-reactive protein (us-CRP), adiponectin, insulin, glucose, aspartate aminotransferase (AST), alanine transaminase (ALT) and gamma-glutamyl transpeptidase (Gamma GT).
Serum selenium was obtained by atomic absorption spectrophotometry (Graphite Furnace) with Zeeman Corrector. For classification purposes, a cut-off point ≤ 45 µg/L was adopted for inadequacy. Glutathione peroxidase activity was measured by the enzymatic method and the MDA was measured in the serum using the colorimetric method in whole blood.
Enzymatic-colorimetric methods evaluated the lipid profile, including the triglyceride (TG), total cholesterol (TC), and HDL-c parameters. LDL-c and VLDL-c were calculated using the formula by Friedewald et al. (1972) [30]. The cut-off points suggested by the American Academy of Pediatrics [31] and the National Cholesterol Education Program (NCEP) [32] were adopted for classification purposes. The presence of dyslipidemia was considered when the TC > 170 mg/dL for children/adolescents and > 200 mg/dL for adults, or LDL-c > 110 mg/dL for children/adolescents and > 129 mg/dL for adults, or triglycerides > 100 mg/dL for children/adolescents and > 150 mg/dL for adults, or HDL-c < 35 mg/dL for children/adolescents, < 40 mg/dL for women and < 50 mg/dL for men.
The values of non-HDLc cholesterol (NHDL-c) were obtained by subtracting the HDL-c values from the TC values and classified according to the work of Bogalusa [33] and NCEP. The following ratios were also calculated: total cholesterol/HDL-c, LDL-c/HDL-c [34], Apo B/Apo A-1, LDL-c/Apo B, and HDL-c/Apo A-1 [35].
Glycemia was measured using a GLUC3/Roche kit (Indianapolis, IN - USA), using an enzymatic reference method with hexokinase, and insulin using an Elecsys Insulin/Roche kit (Indianapolis, IN-USA), using electrochemiluminescence. The fasting blood glucose and insulin values calculated the HOMA-IR (Homeostasis Model Assessment of Insulin Resistance) using the following formula: HOMA-IR = fasting glucose (mmol/l) x fasting insulinemia (µU/ml)/22.5. HOMA-IR was considered changed > 3.16 [36].
Alanine transaminase was measured by an ALTL/Roche kit (Indianapolis, IN-USA), using the enzymatic-kinetic method; aspartate aminotransferase by ASTL/Roche kit (Indianapolis, IN-USA), using the enzymatic-kinetic method, and the gamma-glutamyl transpeptidase range by the GGT2/Roche kit (Indianapolis, IN-USA), using the enzymatic colorimetric method. The complete blood count was performed in whole blood using the cytochemical/isovolumetric method.
The dry tubes directed to the Clinical Analysis Laboratory of the ABC Medical School were centrifuged at 10,000 rpm for 10 minutes. The serums obtained after centrifugation were stored at -80 °C and used to determine the non-classic markers of lipid metabolism: quantity and quality of Apo A-1, Apo B, LDLox and of inflammation biomarkers: us-CRP and adiponectin. All analyses were performed in duplicate. These biomarkers have no benchmark values in the pediatric age group.
The determination of Apo A-1 was performed using the Apo A-1 ver.2/Roche kit (Indianapolis, IN-USA) by the turbidimetry method; that of Apo B, by the Apo B kit ver. 2/Roche (Indianapolis, IN-USA) using the turbidimetry method; that of oxidized LDL, by the Human OxLDL/Wuhan Fine Biological Technology Co kit (Wuhan, China), by the ELISA method.
The CRPLX/Roche kit (Indianapolis, IN-USA) measured the ultrasensitive C-reactive protein using the turbidimetry method. Adiponectin was measured by the Adiponectin Human ELISA kit/ABCAM kit (San Francisco, USA), with the ELISA method. The cutoff point used to indicate elevation was us-CRP ≥ 8.
Statistical Analysis
The SPSS 25.0 (IBM®) program was used for statistical analysis. Categorical variables were shown as absolute numbers and percentages, compared using the Chi-square test. The continuous variables were evidenced in the median and interquartile range and compared using the Mann-Whitney test. The Spearman test was used to assess the correlation between continuous variables and glutathione peroxidase and selenium levels.