Riboflavin (vitamin B2) is crucial for energy metabolism and several physiological processes. Pregnant women have a greater chance of being riboflavin-deficient, but its effects on offspring development remain understudied. Two-month-old female Wistar (WNIN) rats were randomly divided into three groups: control (AIN-93G diet with 5 mg/kg riboflavin), pair-fed, and riboflavin-deficient (0.5 mg/kg). After 6 weeks, riboflavin deficiency was confirmed in the riboflavin-deficient group. All groups of rats were allowed to mate with male littermates. The pups stayed with their mothers during lactation and received a diet similar to the mothers' during weaning. Four pup groups were studied: control, pair-fed, deficient, and replenished. Blood FAD levels were estimated using HPLC. Reproductive indices in dams, developmental milestones, and morphometric data in pups were assessed. Pups were maintained for 6 weeks (postnatal day 42) before being sacrificed. Adult female rats fed a riboflavin-deficient diet showed decreased body weight. While maternal fertility and pregnancy indices were unaffected, litter size was reduced in the riboflavin-deficient group of dams. Pups born to deficient dams exhibited delayed milestones, including pinna unfolding, eye slit formation, and fur coat formation. Morphometric measures, such as body weight, body length, and abdominal circumference, were significantly lower. Post-weaning mortality was higher in the deficient group, likely due to hypoglycemia. Replenishment with a riboflavin-sufficient diet partially improved the outcomes. Maternal riboflavin deficiency impairs offspring growth and developmental milestones. Adequate maternal riboflavin intake is critical for ensuring optimal fetal and postnatal development.

Riboflavin deficiency (RD) induces liver damage, abnormal embryonic development, and high mortality. We hypothesized that the phenotype could be rescued by inhibiting ER stress. The objectives of the present study were to investigate the underlying molecular mechanisms of RD-induced embryonic defects using in vitro and in vivo models. Primary duck embryonic hepatocytes were treated with an ER stress inhibitor (4-PBA) or transfected with CHOP siRNA, and cultured in RD medium and riboflavin-sufficient (CON) medium for 8 days. Laying ducks (n = 20 cages/diet, 1 bird/cage) were fed an RD diet or CON diet for 14 wk, and the eggs were collected for hatching. At day 7 of incubation, the fertilized RD eggs were injected with or without 4-PBA into the yolk. RD decreased cell number and cell viability compared to the CON group, induced oxidative stress and apoptosis in primary duck embryonic hepatocytes. However, after being treated with an ER stress inhibitor (4-PBA) or transfected with CHOP siRNA, the apoptosis rate in RD hepatocytes decreased by 60.6 % and 86.1 %, respectively, being equal to the CON. These results indicated that RD-induced hepatocyte apoptosis is mediated by ER stress and the CHOP pathway. In vivo, RD embryos showed low hatchability, abnormal development, liver damage, ER stress, and apoptosis compared to the CON group. However, 4-PBA administration, as a model of ER stress inhibition, substantially restored embryonic development and alleviated liver damage in the RD group, including ER stress and apoptosis. Notably, hatchability in the RD group increased from 21.7 % to 72.7 % after 4-PBA treatment, though it remained less than the CON group (87.7 %). These results implicated ER stress-CHOP-apoptosis pathway as molecular mechanisms underlying RD-induced abnormal embryonic development and death, this target with potential for therapy or intervention.

Early detection of congenital disorders by newborn screening (NBS) programs is essential to prevent or limit disease manifestation in affected neonates. These programs balance between the detection of the highest number of true cases and the lowest number of false-positives. In this case report, we describe four unrelated cases with a false-positive NBS result for very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Three neonates presented with decreased but not deficient VLCAD enzyme activity and two of them carried a single heterozygous ACADVL c.1844G>A mutation. Initial biochemical investigations after positive NBS referral in these infants revealed acylcarnitine and organic acid profiles resembling those seen in multiple acyl-CoA dehydrogenase deficiency (MADD). Genetic analysis did not reveal any pathogenic mutations in the genes encoding the electron transfer flavoprotein (ETF alpha and beta subunits) nor in ETF dehydrogenase. Subsequent further diagnostics revealed decreased levels of riboflavin in the newborns and oral riboflavin administration normalized the MADD-like biochemical profiles. During pregnancy, the mothers followed a vegan, vegetarian or lactose-free diet which probably caused alimentary riboflavin deficiency in the neonates. This report demonstrates that a secondary (alimentary) maternal riboflavin deficiency in combination with reduced VLCAD activity in the newborns can result in an abnormal VLCADD/MADD acylcarnitine profile and can cause false-positive NBS. We hypothesize that maternal riboflavin deficiency contributed to the false-positive VLCADD neonatal screening results.

Maternal riboflavin deficiency (RD) induces embryonic death in poultry. The underlying mechanisms, however, remain to be established and an overview of molecular alterations at the protein level is still lacking. We investigated embryonic hepatic proteome changes induced by maternal RD to explain embryonic death. A total of 80 45-week-old breeding female ducks were divided into two groups of 40 birds each, and all birds were raised individually for 8 weeks. All the female ducks received either a RD or a riboflavin adequate (control, CON) diet, which supplemented the basal diet with 0 or 10 mg riboflavin /kg of diet respectively. The riboflavin concentrations of maternal plasma and egg yolk, as well as egg hatchability declined markedly in the RD group compared to those in the CON group after 2 weeks, and declined further over time. The hepatic proteome of E13 viable embryos from 8-week fertile eggs showed that 223 proteins were upregulated and 366 proteins were downregulated (> 1.5-fold change) in the RD group compared to those in the CON group. Pathway analysis showed that differentially expressed proteins were mainly enriched in the fatty acid beta-oxidation, electron transport chain (ETC), and tricarboxylic acid (TCA) cycle. Specifically, all the proteins involved in the fatty acid beta-oxidation and ETC, as well as six out of seven proteins involved in the TCA cycle, were diminished in the RD group, indicating that these processes could be impaired by RD. Maternal RD leads to embryonic death of offspring and is associated with impaired energy generation processes, indicated by a number of downregulated proteins involved in the fatty acid beta-oxidation, ETC, and TCA cycle in the hepatic of duck embryos. These findings contribute to our understanding of the mechanisms of liver metabolic disorders due to maternal RD.