The impact of dietary adherence and formula intake regularity on fat-soluble vitamin status in phenylketonuria (PKU) is uncertain. This study assessed whether vitamin A, D, E, and beta-carotene levels differ by dietary adherence and regularity of Phe-free formula intake.

A cross-sectional study included 98 individuals (age 6–41 years) with vitamin D measurements. In a subgroup of 68 patients, vitamin A, vitamin E, and beta-carotene levels were determined. Vitamin levels were compared between adherent and non-adherent groups and between participants with regular vs. irregular formula intake. A subsequent systematic review and meta-analysis of six studies (from PubMed, Scopus, Web of Science, and Cochrane; searched in August 2025) pooled standardised mean differences (SMDs) using fixed-effects and random-effects models.

The cross-sectional results showed higher vitamin D in adherent (35.60 [30.39–41.65] vs. 32.90 [26.50–40.00] ng/mL, p = 0.034) and regular formula consumers (35.97 [30.03–42.28] vs. 30.20 [26.08–35.06] ng/mL, p = 0.002). Beta-carotene was elevated with regular intake (74.40 [56.70–98.45] vs. 53.20 [34.10–68.60] ng/mL, p = 0.003). Meta-analysis confirmed higher vitamin D in adherent individuals (fixed-effects model, SMD = 0.290, 95% CI: 0.004, 0.576, p = 0.047) and regular consumers (fixed-effects model, SMD = 0.750, 95% CI: 0.382, 1.118, p < 0.0001). No differences were observed for vitamin E or beta-carotene.

Adherence to diet and regular formula intake is associated with improved vitamin D status, underscoring the critical role of fortified formulas in PKU management. The very low certainty of evidence necessitates further research, especially for the other fat-soluble vitamins. Nonetheless, clinical practice should emphasise support for adherence and ongoing nutritional monitoring.

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by mutations in the gene encoding phenylalanine (Phe) hydroxylase, the hepatic enzyme responsible for converting Phe to tyrosine. In the absence of adequate enzyme activity, Phe accumulates to neurotoxic levels, leading to irreversible intellectual disability and other neurological impairments if untreated. Early diagnosis through newborn screening and immediate initiation of dietary therapy have dramatically improved clinical outcomes. Managing PKU requires lifelong adherence to a Phe-restricted diet and the use of specialised formulas fortified with essential micronutrients.

However, commitment to the treatment varies widely among patients of all ages, influencing metabolic control and nutritional status. The complexity of PKU management and social burden can lead to poor adherence—particularly during adolescence and adulthood. As individuals transition toward independence, they tend to reduce or discontinue the use of metabolic formulas and increase their intake of Phe-rich foods, resulting in poor metabolic control and potential nutritional imbalances. In this context, attention has increasingly focused on the adequacy of micronutrient intake in PKU. The exclusion of high-protein, nutrient-dense foods, combined with variable adherence to fortified medical formulas, creates a risk of various deficiencies. Fat-soluble vitamins such as A, D, and E, as well as beta-carotene, are of particular interest due to their essential physiological roles and their absorption, which is influenced by dietary composition.

Vitamin A is not a single compound but a group of fat-soluble nutrients that includes two primary forms: preformed vitamin A (retinol, retinal, retinoic acid) and provitamin A carotenoids (beta-carotene, alpha-carotene, and beta-cryptoxanthin). The retinoids are primarily found in animal foods such as beef liver, eggs, and dairy products. In contrast, carotenoids are abundant in plant foods like sweet potato, pumpkin, carrots, apricots, and leafy green vegetables. Once consumed, beta-carotene is converted in the intestinal mucosa by beta-carotene dioxygenase and subsequently reduced to retinol, the active form of vitamin A in the body, which modulates immune responses, supports normal reproductive and visual functions, and regulates cellular proliferation and differentiation. Vitamin A compounds are essential for the proper development and maintenance of vital organs such as the heart, lungs, eyes, and reproductive system. In addition, beta-carotene also functions as an antioxidant, helping to neutralise reactive oxygen species and protect cells from oxidative damage. Reduced plasma beta-carotene in PKU has been reported to correlate negatively with serum Phe concentrations, suggesting that poorer metabolic control is associated with lower circulating carotenoid levels. Evaluating beta-carotene alongside other vitamins may provide additional insight into antioxidant capacity and fat-soluble vitamin metabolism in individuals with PKU.

Vitamin E refers to a group of fat-soluble compounds, primarily tocopherols and tocotrienols. Among them, alpha-tocopherol is the most biologically active and the predominant form in human tissues. Rich dietary sources include nuts, seeds, and vegetable oils such as sunflower, safflower, soybean, and wheat germ oil. The vitamin is also found in legumes, butter, tomatoes, and green leafy vegetables. Vitamin E plays a key role in protecting cell membranes from oxidative damage by inhibiting lipid peroxidation mediated by free radicals. Additionally, it stabilises the cell membranes by interacting with destabilising molecules, modulates enzymatic activity, cell signalling, proliferation, and gene expression, and inhibits platelet aggregation.

Beyond the restricted intake of many natural vitamin-rich, high-protein sources, some studies have reported evidence of oxidative stress in PKU, including increased markers of lipid, protein, and DNA oxidation, as well as compromised antioxidant defences. Under such conditions, lipophilic antioxidants such as alpha-tocopherol may be utilised more intensively to protect cell membranes from peroxidation, potentially lowering circulating vitamin E levels. Previous studies investigating fat-soluble vitamin status in individuals with PKU have shown variable results. Kose et al. reported higher frequencies of elevated alpha-tocopherol levels among adherent patients than among non-adherent patients (21.9% vs. 8.5%), but no differences in beta-carotene or alpha-tocopherol levels between groups. In contrast, Schulpis et al. found that patients adhering strictly to dietary treatment had significantly higher blood levels of beta-carotene and alpha-tocopherol, along with increased antioxidant status. Colome et al. found suboptimal alpha-tocopherol concentrations in 17.2% of 58 PKU patients. Mikoluc et al. observed that retinol levels remained within normal range despite low intake. However, the latter two studies did not consider adherence.

Vitamin D is obtained from limited dietary sources and from cutaneous synthesis via sunlight exposure, with supplementation being an ordinary and often necessary contributor to status. It is initially inactive and requires two hydroxylation steps to become biologically active. The first occurs in the liver, producing 25-hydroxyvitamin D (25(OH)D), and the second in the kidneys, forming the active hormone 1,25-dihydroxyvitamin D (1,25(OH)₂D). Vitamin D is naturally present in fatty fish (e.g., salmon, mackerel), fish liver oils, egg yolks, beef liver, dairy products, and some mushrooms. Beyond its well-established role in calcium metabolism and bone health, vitamin D also contributes to immune regulation , metabolism , hematopoietic cell differentiation, and reproductive function. Emerging evidence links adequate vitamin D levels to reduced risks of certain cancers, including prostate and breast cancer, and to potential benefits in managing mood disorders such as depression and anxiety. Since individuals with classical PKU may be prone to excess weight due to their specific dietary pattern and use of highly processed low-protein foods, increased adiposity may lower circulating 25(OH)D and contribute to the overall risk of vitamin D deficiency in this population. Vitamin D deficiency is common among individuals with PKU. However, it has been documented that levels are higher than those in the general population. Kose et al. identified 25(OH)D deficiency in 53.6% of 112 patients with PKU. In contrast, Silva et al. reported a lower prevalence of 30.6% in a retrospective study of 90 subjects. Rojas-Agurto et al. observed significantly lower serum vitamin D levels in PKU patients who transitioned from Phe-free protein substitutes to primarily vegan diets compared with those who continued supplementation. In contrast, Hochuli et al. found no differences in 25(OH)D levels between patients with adequate vs. suboptimal amino acid mixture intake.

According to a recent review on nutritional management in PKU, in this disorder, strict low-protein diets and reliance on specialised medical foods narrow the range of nutrients reaching the gut, thereby reshaping the gut microbiota and reducing its diversity. This diet-induced dysbiosis, together with altered production of microbial metabolites, such as short-chain fatty acids, and interactions with nutrient supplements, may have lasting effects on gut health, immune function, and overall metabolism.

Our previous systematic review and meta-analysis indicated that individuals with PKU generally maintain adequate levels of vitamins A, E, and 25(OH)D, comparable to those of healthy controls; however, the impact of dietary adherence and Phe-free formula use on fat-soluble vitamin status remains unclear. To address this gap, the present cross-sectional study evaluates serum vitamins A, D, E and beta-carotene in individuals with PKU, stratified by adherence patterns defined using two criteria: (1) metabolic control (mean annual Phe concentrations) and (2) regularity of prescribed Phe-free formula intake. A systematic review and meta-analysis were conducted to synthesise and assess the available evidence on this topic. We hypothesised that vitamin A, D, E, and beta-carotene levels would not differ significantly between patients with lower versus higher Phe levels, nor between those with regular versus irregular use of Phe-free formula.