Micronutrient malnutrition is prevalent in many developing countries and may contribute to a weakening of immune status and thus a worsening of clinical condition in individuals infected with HIV. Studies of various designs and using different measures of clinical or immunological status have been conducted in several geographic locations to examine the importance of vitamins. The results of several trials among adults have confirmed the potential benefits of the B vitamins, including niacin, in immune status among individuals infected with HIV. However, few published data provide information regarding niacin status in HIV-positive children.
Recently, it has been shown in cell culture models that HIV infection induces a state of pellagra, characterized by decreased intracellular nicotinamide, which is reversed by the administration of nicotinamide (1). Pellagra is caused by a diet deficient in niacin (the generic name for nicotinic acid and nicotinamide) and tryptophan, or by an inability to absorb and process these nutrients. Metabolism of dietary tryptophan, via the kynurenine pathway, leads to the formation of niacin (2). Zinc-, iron-, riboflavin-, and vitamin B6-dependent enzymes are involved in the pathway and deficiencies in these nutrients may also contribute to pellagra. Although currently pellagra is rare, mainly because of awareness of it and fortification of food, there is increasing interest in niacin status because of its possible association with HIV-related conditions (3–5). Clinical signs are apparent only when the deficiency is well advanced. The signs include changes to the skin, gastrointestinal tract, and nervous system. The niacin nutritional status of adult patients with AIDS who experienced diarrhea was similar to that of HIV-negative control individuals with pellagra (3).
Nicotinamide adenine dinucleotide is involved in ATP production, and nicotinamide adenine dinucleotide turnover can be investigated by the urinary outputs of its major metabolites, N1-methylnicotinamide (MNA) and N1-methyl-2-pyridone-5-carboxamide (6). Nicotinamide adenine dinucleotide is required in a variety of mechanisms involving energy metabolism and growth. The growth of HIV-positive children may be delayed during niacin deficiency, but not enough evidence is available to serve as a guide in ascertaining the level of intake below which nicotinic acid deficiency may be expected to occur, or the level of urinary MNA excretion that represents niacin deficiency.
Cross-sectional studies in children have indicated that biochemical micronutrient deficiencies are more prevalent in infected children than in uninfected children (7,8), but until now there have been no studies concerning niacin, to our knowledge.
We believe that despite socioeconomic conditions in Brazil, HIV status can be a risk factor for niacin deficiency. The aim of this study was to compare the nutritional status and the 24-hour urine excretion of MNA among HIV-positive children and HIV-negative children who were and were not born to mothers with HIV-1 infection.
PATIENTS AND METHODS
This preliminary cross-sectional comparative study was carried out at the teaching hospital of the School of Medicine of Ribeirão Preto, University of São Paulo, Brazil. Every consecutive child who came to the city reference Pediatric Outpatient Unit in August 2005 was recruited. The institutional ethics committee approved the research protocol, and all of the patients or their parents gave written consent to participate after a detailed explanation to each family. Forty patients, subdivided into 3 groups, were allocated to participate in the study: HIV-positive children (group 1, n = 20), HIV-negative children born to infected mothers (group 2, n = 10), and HIV-negative control children (group 3, n = 10). Inclusion criteria were ability to complete the anthropometry and absence of liver disease, chronic or acute pancreatitis, renal failure, or diarrhea that could interfere in the development of the child and its nutritional status. No patient was receiving vitamin supplementation, appetite stimulants, or enteral or parenteral nutrition.
The HIV-negative children were selected from families of HIV-positive children to avoid socioeconomic and cultural differences. Clinical and demographic data were obtained from charts. HIV infection was determined by enzyme-linked immunosorbent assay and confirmed by Western blot technique.
Nutrient Intake Assessment
Usual dietary intake was assessed by an adapted semiquantitative food-frequency questionnaire (9) according to the method of Margetts et al (10), and intakes of niacin, tryptophan, zinc, vitamin B6, and energy were calculated by use of CIS EPM software. The calculated daily energy expenditure and the adequacy of niacin intake were based on the daily recommended intake (DRI) (11,12).
Body Composition Assessment
Weight and height were assessed according to the method of Heymsfield et al (13) by a dietitian who was trained to take all measurements. The reference data of the World Health Organization/National Center for Health Statistics/Centers for Disease Control and Prevention (14) was used. For the estimation of fat-free mass and total body water, a bioelectrical impedance technique was applied at the same time the weight was measured (15). The midarm circumference, subscapular skinfold, and triceps skinfold were also measured to reflect lean body mass and fat (16). The nutritional status of all of the children was classified according to Waterlow (17).
To confirm the diagnosis of niacin deficiency, urine specimens were taken (24-hour samples) from all of the patients. Urine samples were collected on the same week as nutritional assessment and were stored at −20°C until analysis. MNA was measured by a modified method of high-performance liquid chromatography (18). Analysis was performed with a Luna C18 (particle size 5 μm, pore diameter 100 Å) 150 mm × 4.6 mm column, protected with a C 18-mm (ODS) 4-mm (L) × 3.0-mm (D) guard cartridge (Phenomenex UK Ltd, Macclesfield, UK) and maintained at 25°C. The sample was resuspended in the mobile phase (15% acetonitrile, 85% water, and 0.15% KH2PO4) and injected into the chromatograph. A fluorometer detector with an emission wavelength of 440 nm and an excitation wavelength of 365 nm was used, and concentrations of MNA in urine were determined. Standard 1-methylnicotinamide chloride was purchased from Sigma-Aldrich (Poole, UK). All of the other chemicals were of analytic grade and were purchased from a variety of suppliers.
All MNA values obtained were expressed in milligrams per gram of creatinine excreted. Concentrations of MNA below 2 mg/g creatinine were considered indicative of niacin deficiency risk. Urinary creatinine was measured by the spectrophotometric methods of Henry et al (19) and Owen et al (20).
Continuous variables with normal distribution were expressed as mean ± standard deviation; in this case, comparison across groups was done by analysis of variance and nonpaired t test. Nonparametric Kruskal-Wallis and Mann-Whitney tests were used to compare variables with non-normal distribution, which were expressed as median and range. The χ2 test or Fisher exact test was used to compare frequency distributions across groups. P < 0.05 was considered statistically significant.
A total of 88 HIV-positive children were initially recruited. After exclusion of those who did not meet the strict inclusion criteria, 29 children were eligible to participate, but written consent was obtained for only 20. Of those 20 HIV-positive children, 15 had HIV-exposed uninfected siblings, but only 10 were matched for sex and age and agreed to participate. The HIV-negative children (n = 10) were also recruited from the HIV-positive children's relatives, mainly cousins living in similar socioeconomic conditions, whose mothers were HIV negative and agreed to participate, and the children were also matched for sex and age.
The groups were matched by age (group 1, 7.85 ± 1.70 years; group 2, 8.35 ± 1.5 years; group 3, 8.33 ± 1.14 years), sex (60% boys in group 1, 60% boys in group 2, and 60% boys in group 3), and percentage of malnutrition (40% in group 1, 30% in group 2, and 40% in group 3). All of the patients in group 1 had a diagnosis of AIDS but were clinically stable, and the basic parameters of immune status as determined by CD4 count and viral load, respectively, were as follows: median = 631.5; range = 172 to 1938 cells/mm3; median = 6542; range = 50–68,496 copies/mL. Except for 3 children, all were receiving antiretroviral therapy. No child had diarrhea, dermatitis, or dementia, and none were using vitamin supplements. Groups were matched in relation to nutritional status parameters, including anthropometric measures and body composition (Table 1).
The food frequency questionnaire was chosen to estimate the usual intake of the previous 3 months (Table 2). The children had similar and adequate daily energy intake. Protein intake was also similar and adequate across group 1, group 2, and group 3. Daily niacin intake did not differ statistically across groups, nor did intake of tryptophan, vitamin B6, or zinc. The mean or median of all of the nutrients met the DRI. Only 22.2% of group 1, 10% of group 2, and 20% of group 3 had niacin intake below the DRI, without statistical differences.
The daily urinary excretion of MNA by all children is shown in Table 2. The median and range values of urinary niacin per gram of creatinine were similar and adequate across the groups. The percentage of children with urinary MNA <2 mg/g creatinine was equal among groups (group 1 = 15%; group 2 = 10%; group 3 = 10%; P > 0.05).
To our knowledge, this is the first study to evaluate niacin status in HIV-positive children, and the results showed that our HIV-positive children excreted the same amount of MNA in urine as did the HIV-negative children born to HIV-positive mothers and the control children. These findings may be attributed to similarities in intake of niacin, tryptophan, zinc, vitamin B6, and protein; adequate intestinal absorption (no patient experienced diarrhea); similarities in anthropometric measures; and similarities in fat-free mass, as measured by bioelectrical impedance analysis.
Although validation studies of energy intake data have led to the widespread recognition that much of the dietary data on children and adolescents is prone to underreporting error (21), we found adequate and similar energy and nutrient intake among the 3 groups. The food frequency questionnaires have been used and validated to assess intakes in many different study populations (22).
Children infected by HIV usually have nutritional problems, but we did not find any differences in body composition and in the anthropometric indices among the 3 groups. Leandro-Merhi et al (23) showed that during the first 2 years of life, the z-scores for infected children regarding weight and length in Brazilian infected children were less than those for noninfected children, but until now, similar studies have not been conducted with older children as far as we are aware. The applicability of the bioelectrical impedance technique pediatric prediction equations for total body water and for fat-free mass to children with specific medical problems has been questioned (24). We believe our data are reliable because we dealt with children in a clinically stable condition, and no HIV-positive children experienced morphological changes attributable to lipodystrophy, including loss of subcutaneous adipose tissue and facial wasting. Furthermore, measures of skinfold thickness, which have been correlated to the bioelectrical impedance technique (25), were similar among groups and in accord with the results observed by Alfaro et al (26). In all of the groups, the average daily intake of niacin and median daily urinary excretion of N1-methylnicotinamide were similar to those observed by Miller and Abernathy (27) in a sample of 7- to 9-year-old healthy children.
Monteiro et al (3) showed that niacin deficiency developed in 100% of adult AIDS patients. Notwithstanding our lack of understanding the mechanism for niacin depletion in HIV infection, in vitro data suggest that nicotinamide, the active intracellular form of niacin, acts as an inhibitor of HIV replication (28) and is depleted by HIV infection (1). In the present study viral load was lower than 10,000 copies/mL in 60% of HIV-positive children, which could explain our results, at least in part.
It could be speculated that the adequate urinary MNA in our HIV-positive children reflected a disease condition rather than a deficiency state (29). Further investigations are needed to confirm this hypothesis. Nevertheless, given that the HIV-positive children had the same food history, the same body composition, and the same anthropometric measures, this may be not relevant.
In conclusion, our preliminary data suggest that our HIV-positive children have a good nutritional status related to niacin, which is probably associated with an adequate nutritional status and to the antiretroviral therapy (30). Such status may confer a potential benefit on immune status among these children. However, long-term investigations with large samples need to be conducted to ensure not only that the weight and height growth of these children can be maintained at the same rate as in uninfected children but also that these children continue to develop mentally, neurologically, and psychologically, appropriately reaching physical, emotional, and sexual maturation.
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