Elsevier

Advanced Drug Delivery Reviews

Volume 55, Issue 11, 14 November 2003, Pages 1467-1483
Advanced Drug Delivery Reviews

Current perspectives on the cellular uptake and trafficking of riboflavin

https://doi.org/10.1016/j.addr.2003.07.005Get rights and content

Abstract

The role of riboflavin in cell maintenance and growth, and the mechanism by which it is absorbed into various human tissues and cell lines has been extensively studied over the past decade. Evidence suggests two absorption mechanisms, a saturable-active component that dominates at near physiological vitamin concentrations and a passive component that is revealed at oversupplemented riboflavin conditions. Various transport modulator studies consistently suggest a highly riboflavin specific, temperature-dependent active transport mechanism that is regulated by the Ca2+/calmodulin pathway. The PKA and PKG pathways have also been implicated in absorption regulation. The long-standing model that riboflavin absorption involves a carrier-mediated transporter has recently been challenged through studies suggesting a receptor-mediated endocytic component. The presence of a soluble, human riboflavin binding protein in the transport stratagem has been shown to play an important role in fetal development. The relationship of this binding protein with the riboflavin specific membrane bound protein, though currently not well defined, may involve a protein–protein interaction that plays a primary role in this proposed receptor-mediated component.

Introduction

Riboflavin (Rf), also known as vitamin B2, is a water-soluble vitamin essential for normal cellular functions, growth and development. In its coenzyme forms of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), Rf performs key metabolic functions as an intermediary in the transfer of electrons in biological oxidation–reduction reactions. During periods of dietary deprivation or physiological and pathological stress, humans are vulnerable to developing Rf deficiency. This may lead to a variety of clinical abnormalities, including growth retardation, anemia, skin lesions and degenerative changes in the nervous system [1]. Humans cannot synthesize Rf and, thus, must obtain the vitamin from their diet through absorption in the small intestine. Alternatively, Rf is also obtained from indigenous bacteria that colonize the large intestine, which naturally synthesize this vitamin. Although many studies have focused on the mechanism of Rf uptake using various tissues and cell lines, transepithelial absorption is controversial and its exact mechanism(s) remains to be defined further. Intracellular processes in Rf absorption, such as cellular homoeostasis, and Rf function and regulation are also poorly understood. The purpose of this review is to illustrate current in vitro and in vivo data defining Rf uptake mechanisms noted in different studies and to compare and contrast the various implicated parameters defining this (these) absorption mechanism(s). In addition, Rf subcellular trafficking events involving receptor-mediated endocytosis and cytoskeletal elements will be discussed. The involvement of a soluble riboflavin binding/carrier protein (RfBP, RCP) in Rf uptake and trafficking is currently elusive. Although several studies have reported its existence [2], [3], it has not been characterized in detail. The majority of the work on RfBP has been focused on oviparous species and has shown a crucial role in embryo development. Few studies have been carried out on mammalian RfBP analogs despite its promising therapeutic applications. This review will consider the similarities in protein sequence and function between RfBP for oviparous and mammalian species. Based on uptake and transport results using RfBP in the presence of Rf, suggested roles in Rf absorption and trafficking events will be presented. Because parallels can be drawn between the uptake and transport mechanisms of Rf and other well-studied vitamins, a brief discussion of commonalities defining such mechanisms for folate will be illustrated. Here the authors aim to present comparisons with current knowledge on Rf uptake and transport, and how these prior studies may guide future elucidation of Rf-specific pathways functioning in Rf homeostasis.

Flavoproteins constitute one of the largest groups of functionally related proteins currently known, catalyzing essential oxidation/reduction steps in almost every metabolic pathway in both prokaryotic and eukaryotic cells. Using essentially the same Rf-based cofactor, flavoproteins participate in a remarkable array of biological processes, from simple electron transfer to complex signal transduction pathways. It is not surprising that defects within critical electron transfer processes may manifest themselves in severe, and sometimes fatal, metabolic diseases. Most dietary Rf is presented in the form of free Rf, FAD, FMN and flavoproteins, which must first be hydrolyzed to Rf before absorption can occur (Fig. 1). Digestion of flavoproteins, FMN and FAD occurs by highly specific enzymes [4], suggesting a physiologic preference that favors Rf as the absorptive species of dietary flavin. Though there is extensive information on the function of mucosal and cytosolic enzymes involved in the digestion of flavoproteins (e.g., flavokinase and FAD synthetase), it is interesting to note that none of these enzymes have been cloned or otherwise structurally characterized.

Therapeutically, Rf has recently gained renewed interest after it was shown to protect vital tissues from ischemia-induced oxidative injury resulting from heart attack or stroke [5], [6]. Given the critical role that Rf appears to play in pathological conditions with high morbidity, it is pertinent for the cellular homeostasis and the pathway by which this vitamin gains entry to the body to be further defined.

In animals, Rf deficiency results in lack of growth, failure to thrive, and eventual death [1]. Experimental riboflavin deficiency in dogs results in growth failure, weakness, ataxia, and inability to stand. The animals collapse, become comatose and die [1]. During the deficiency state, dermatitis develops together with hair-loss. Other signs include corneal opacity, lenticular cataracts, hemorrhagic adrenals, fatty degeneration of the kidney and liver, and inflammation of the mucus membrane of the gastrointestinal tract [7]. Post-mortem studies in rhesus monkeys fed a Rf deficient diet revealed that about one-third the normal amount of Rf was present in the liver [8], which is the main storage organ for Rf. These overt clinical signs of Rf deficiency are rarely seen among inhabitants of the developed countries. However, about 28 million Americans exhibit a common ‘subclinical’ stage [9], characterized by a change in biochemical indices (e.g., erythrocyte glutathione reductase [10]). Although the effects of long-term subclinical Rf deficiency are unknown, in children this deficiency results in reduced growth. Subclinical Rf deficiency has also been observed in women taking oral contraceptives [11], in the elderly, eating disorders, and in disease states such as HIV [12], inflammatory bowel disease [13], diabetes and chronic heart disease [1]. The fact that Rf-deficiency does not immediately lead to gross clinical manifestations indicates that the systemic levels of this essential vitamin are tightly regulated.

Section snippets

Riboflavin uptake mechanism

Between the early 1940s and late 1970s, numerous studies described riboflavin pharmacokinetics and pharmacodynamics in man and other mammalian species. These initial studies provided the foundation of data suggesting the existence of a specialized Rf absorption mechanism [14]. Jusko and colleagues [15] demonstrated in man a significant inhibition in renal clearance of Rf upon combined treatment with probenecid, now known to be a potent inhibitor of many organic anion transport systems. The same

Receptor-mediated or carrier-mediated transport?

Up until recently, it was generally accepted that Rf translocation followed an active, carrier-mediated pathway. However, the criteria used to define active transport, i.e. energy dependency and saturation transport kinetics, do not effectively distinguish carrier-mediated transport from receptor-mediated endocytosis (RME). Receptor-mediated events typically involve endocytosis followed by microtubule driven vesicular sorting to various cellular organelles [36]. In an effort to determine

Riboflavin binding/carrier protein

Plasma protein binding and translocation of riboflavin in mammals are non-specific under normal conditions. During pregnancy, this non-specific transport by albumin and riboflavin binding immunoglobulins is not adequate to provide the developing fetus with sufficient Rf for normal development and growth. It is believed that under these demanding conditions specific carrier proteins are expressed that deliver riboflavin to the developing embryo. These soluble plasma proteins are referred to as

Comparison with folate uptake and transport mechanism

RfBP belongs to the family of folate binding proteins (FBP) that are glycosylphosphatidylinositol (GPI) anchored proteins and found on the outer surface of the plasma membrane. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in milk FBP, as are all of the tryptophan residues [53]. This conservation of cysteine residues indicates the importance of secondary structure and function of RfBP. Sequence identity between homologous regions of these two

Conclusion

Over the last decade, our understanding of the molecular regulation and cellular transport mechanism of Rf has significantly increased. In general, Rf absorption exhibits the following biochemical properties: temperature dependence, a general Na+-independence, a saturable active transport component that appears to dominate at near basal Rf concentrations and a passive diffusion component that dominates at concentrations above physiological Rf concentrations. In addition, the Ca+/calmodulin-,

Acknowledgements

This work was supported by a grant from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK56631).

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