Studies on the transport of aliphatic glucosides by hamster small intestine in vitro.

It has been found (1) that glucosides with a long alkyl chain (2-18 carbon atoms) as the aglycone can be transported by carrier-mediated processes in the hamster small intestine in vitro, (2) that these glucosides interact with the glucose carrier, and (3) that they compete with glucose and analogs for the binding to the carrier. The are Na+- and phlorizin-insensitive components of uptake for the long chain alkyl glucosides which suggest additional interactions or uptake processes.     

C-terminus loop 13 of Na+ glucose cotransporter SGLT1 contains a binding site for alkyl glucosides

Recently, we identified the extramembranous C-terminus loop 13 of SGLT1 as a binding site for the aromatic glucoside phlorizin, which competitively inhibits sodium D-glucose cotransport. Alkyl glucosides are also competitive inhibitors of the transport. Therefore, in this study, we searched for potential binding sites for alkyl glucosides in loop 13. To this end, we synthesized a photoaffinity label (2'-Azi-n-octyl)-beta-D-glucoside and analyzed the region of attachment using MALDI mass spectrometry, producing wild-type recombinant truncated loop 13. Furthermore, we prepared four single-Trp mutants of the loop and determined their fluorescence, its change in the presence of alkyl glucosides, and their accessibility to acrylamide. Photolabeling of truncated loop 13 with (2'-Azi-n-octyl)-beta-D-glucoside revealed an attachment of the C2 group of the alkyl chain to Gly-Phe-Phe-Arg (amino acid residues 598-601). In the presence of n-hexyl-beta-D-glucoside, all mutants (R601W, D611W, E621W, and L630W) exhibited a significant decrease in Trp fluorescence with an apparent binding affinity of 8-14 microM. Only L630W exhibited a significant blue shift, and only in R601W was a change in acrylamide quenching (protection) observed. No quenching or protection was found for D-glucose; however, 1-hexanol produced the same results as n-hexyl-beta-D-glucoside. The interaction shows stereoselectivity for n-hexyl-beta-D-glucoside binding; the beta-configuration of the sugar moiety at C1, the cis conformation of the unsaturated alkenyl side chain in the C3-C4 bond, and the alkyl chain length of six to eight carbon atoms lead to an optimum interaction. A schematic two-dimensional model was derived in which C2 interacts with the region around residue 601, C3 and C4 interact with the region between residues 614 and 619, and C6-C8 interact with the region between residues 621 and 630. The data demonstrate that loop 13 provides binding sites for alkyl glucosides as well as for phlorizin; thus, loop 13 of SGLT1 seems to be a major binding domain for the aglucone residues of competitive D-glucose transport inhibitors.     

Competition between transglycosylation and hydrolysis in almond β-glucosidase-catalyzed conversion of p-nitrophenyl-β-d-glucoside in monophasic water/alcohol mixtures

Almond β-glucosidase was used to catalyze the transglycosylation of p-nitrophenyl-β-d-glucoside to alkyl glucosides, with hydrolysis to glucose as a side reaction. The conversions were carried out in alcohols with varying water contents below water saturation. Both the total reaction rate and the ratio between the transglycosylation and hydrolysis increased with increasing water activity, and at a fixed water activity in the different alcohols, rate and transglycosylation/hydrolysis ratio increased in the following order: 1-octanol<1-hexanol<1-butanol. Synthesis of alkyl glucosides by transglycosylation in monophasic alcohol media is thus most favorable for short chain alcohols, and should be carried out at high water content.     

Saturable uptake of indol-3yl-acetic Acid by maize roots

The uptake of 5-[³H]indol-3yl-acetic acid (IAA^*) by segments of Zea mays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA°) at different concentrations. IAA uptake was found to have a nonsaturable component and a saturable part with (at pH 5.0) an apparent K_m of 0.285 micromolar and apparent V_max 55.0 picomoles per gram fresh mass per minute. These results are consistent with those which might be expected for a saturable carrier capable of regulating IAA levels. High performance liquid chromatography analyses showed that very little metabolism of IAA^* took place during 4 minute uptake experiments. Whereas nonsaturable uptake was similar for all 2 millimeter long segments prepared within the 2 to 10 millimeter region, saturable uptake was greatest for the 2 to 4 millimeter region. High levels of uptake by stelar (as compared with cortical) segments are partly attributable to the saturable carrier, and also to a high level of uptake by nonsaturable processes. The carrier may play an essential role in controlling IAA levels in maize roots, especially the accumulation of IAA in the apical region. The increase in saturable uptake toward the root tip may also contribute to the acropetal polarity of auxin transport.     

Transglucosylation of tertiary alcohols using cassava beta-glucosidase

We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from p-nitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis.     

Destruction of highly purified cytochromes P-450 associated with metabolism of fluorinated ether anesthetics

The effects of ethanol and acetaldehyde on rat intestinal microvillus membrane integrity and glucose transport function were examined in vitro with purified membrane vesicles. Ethanol could influence glucose transport function by alterations in the conformation of the carrier, the lipid environment surrounding the carrier, or in the transport driving force (Na⁺ electrochemical gradient). Due to the rapid nature of glucose uptake, transport was assayed with the use of an apparatus that permitted uptake measurements as early as 1 s. Ethanol (340 mm) partially and acetaldehyde (44 mm) completely inhibited concentrative glucose uptake throughout the 1-min time course. Their inhibitory effects were reversible and irreversible, respectively. Kinetic measurements made during the initial rate of uptake (at 2 s) with various concentrations of glucose (0.05–8 mm) showed that ethanol and acetaldehyde both caused a decrease in V. Although ethanol did not substantially alter the transport K_m, acetaldehyde increased the K_m almost 50%. To determine whether ethanol or acetaldehyde directly interfered with glucose carrier function, uptake was measured in the presence of equilibrated Na⁺. Only acetaldehyde had a significant inhibitory effect under these conditions. Membrane permeability, as determined by efflux of preloaded 6-carboxyfluorescein dye, increased upon exposure of the vesicles to ethanol or acetaldehyde. Membrane fluidity measurements by fluorescence polarization showed that only acetaldehyde had a significant fluidizing effect. These results indicate that ethanol and acetaldehyde acted to perturb membrane integrity and inhibited glucose uptake indirectly by allowing the Na⁺ gradient to dissipate. Acetaldehyde, which had a stronger inhibitory effect than ethanol, appeared also to directly inhibit carrier function.     

Crystal structures and thermal analyses of alkyl 2-deoxy-α-d-arabino-hexopyranosides

The crystal structures of alkyl 2-deoxy-α-d-arabino-hexopyranosides, with the alkyl chain lengths from C₈ to C₁₈, are established by the single crystal X-ray structural determination. The even-alkyl chain length derivatives crystallized orthorhombic, with space group P2₁2₁2₁, whereas the odd-alkyl chain length derivatives crystallized monoclinic, with space group P2₁. The sugar moieties retained a ⁴C₁ chair conformation and the conformation of the alkyl chains was all-trans. The molecules formed a bilayer structure, in which alkyl chains were interdigitated. The hydrogen bonds, originating from the sugar moieties, were observed in adjacent layers and also within the same layer, resulting in the formation of infinite chains. The alkyl chains arranged parallel to each other and formed planar structures. The thermal properties of the alkyl 2-deoxy glucosides were analyzed further. It was observed that none of the derivatives exhibited mesomorphism. This study establishes that the absence of the hydroxyl group at C-2 of the sugar moiety results in a non-mesogenic nature of the alkyl 2-deoxy-α-d-glycosides, as opposed to the profound mesogenic nature of the normal alkyl glycosides.     

Specificity of the Glucose Transport System in Leishmania tropica Promastigotes*

SYNOPSIS. The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q₁₀ of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (α-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3–0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature—they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident, therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Changes in the C-Labeled Cell Wall Components with Chase Time after Incorporation of UDP[C]Glucose by Intact Cotton Fibers

Intact, in vitro-grown cotton fibers will incorporate [¹⁴C]glucose from externally supplied UDP[¹⁴C]glucose into a variety of cell wall components including cellulose; this labeled fraction will continue to increase up to 4 hours chase time. In the fraction soluble in hot water there was no significant change in total label; however, the largest fraction after the 30 minute pulse with UDP[¹⁴C]glucose was chloroform-methanol soluble (70%) and showed a significant decrease with chase. The lipids that make up about 85% of this fraction were identified by TLC as steryl glucosides, acylated steryl glucosides, and glucosyl-phosphoryl-polyprenol. Following the pulse, the loss of label from acylated steryl glucosides and glucosylphophoryl-polyprenol was almost complete within 2 hours of chase; steryl glucosides made up about 85% of the fraction at that chase time. The total loss in the lipid fraction (about 100 picomoles per milligram dry weight of fiber) with chase times of 4 hours approximates the total gain in the total glucans.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca²⁺ uptake by brain tissue, suggesting a relationship between glucose and Ca²⁺ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca²⁺ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca²⁺ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca²⁺ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca²⁺ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca²⁺ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca²⁺ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca²⁺ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca²⁺ channels, did not alter Ca²⁺ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca²⁺ uptake was not mediated by Ca²⁺ channels. Tetrodotoxin which specifically blocks Na⁺ channels, abolished Ca²⁺ uptake enhanced by glucose deprivation, but had no effect on Ca²⁺ uptake in presence of glucose (controls). These results suggest that stimulation of Ca²⁺ uptake by glucose deprivation may be related to Na⁺ transfer via Na-Ca exchange in brain.     

Effects of the Fenton reagent on transport in yeast

In the facultatively anaerobic yeastSaccharomyces cerevisiae the uptake rate and the accumulation ratio of 2-aminoisobutyric acid was decreased by some 30% by Fenton's reagent (FR), a powerful source of OH… radicals. Likewise, the uptake of glutamic acid, leucine and arginine was diminished. The mediated diffusion of 6-deoxy-d-glucose was not affected. The H⁺ symport of maltose and trehalose was inhibited by some 40% both in the initial rate and in the accumulation ratio. FR had a dramatic inhibitory effect when present during preincubation with 50 mmol/L glucose. In the obligately aerobicLodderomyces elongisporus the uptake of all amino acids tested was decreased by 15–30%, that of 6-deoxy-d-glucose by about 10%. The initial rates of uptake of maltose and trehalose were depressed by FR by 40% and the acceleration of uptake observed after 8 min of incubation, was abolished by FR completely. Acidification rate of the external medium byS. cerevisiae in the presence of glucose or galactose was enhanced three-fold, that after subsequently added K⁺ was substantially decreased. FR appears to have a dual effect on sugar and amino acid transport processes in yeast: (1) it blocks carrier protein synthesis, (2) it inhibits the source of energy for transport. It does not appreciably affect the carrier proteins themselves.     

Isolation of a cytosolic beta-glucosidase from calf liver and characterization of its active site with alkyl glucosides and basic glycosyl derivatives

A beta-glucosidase/beta-galactosidase with Mr 52,500 was isolated from calf liver cytosol by a four-step procedure incorporating affinity chromatography on N-(9-carboxynonyl)-deoxynojirimycin-AH-Sepharose. Its pH optimum was at 5.8 with half-maximal activity at pH 3.5 and 8.6. Affinity for gluco compounds expressed by Km or Ki of substrates and inhibitors was 2- to 10-fold higher than for the corresponding galacto compounds. Alkyl glucosides were hydrolyzed with lower Vmax than p-nitrophenyl and 4-methylumbelliferyl glucosides, but due to their higher affinity the alkyl glucosides displayed values for kcat/Km of the same magnitude of the aryl glucosides when the alkyl chains were longer than octyl. Glucosylsphingosine was bound with Ki (= Km) 2.2 microM and hydrolyzed with a Vmax that was 50-fold lower than the Vmax for 4-methylumbelliferyl beta-glucoside. The product sphingosine was inhibitory with Ki 0.30 microM. A systematic study with alkyl glucosides and glucosylamines defined the aglycon site as a narrow, strongly hydrophobic cleft able to accommodate up to 10 methylene groups. Each CH2 group contributed 3.1 kJ/mol to the standard free energy of binding. The inhibition by gluco- and galactosylamine and by 1-deoxynojirimycin and its D-galacto analog was approximately 200-fold better than by corresponding nonbasic compounds. pH dependence of the inhibition and comparison with permanently cationic glycosyl derivatives showed that the nonprotonated form was the inhibiting species. This feature puts the cytosolic beta-glucosidase in the large class of glycoside hydrolases which strongly bind basic glycosyl derivatives by their protonation at the active site and formation of a shielded ion pair with the carboxylate of an aspartic or glutamic side chain.     

Block of the Kir2.1 Channel Pore by Alkylamine Analogues of Endogenous Polyamines

Inward rectification induced by mono- and diaminoalkane application to inside-out membrane patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines, increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a limiting minimal value of ∼1.5 (n = 2 methylene groups) to ∼4 (n = 10 methylene groups). These observations lead us to hypothesize that monoamines and diamines block inward rectifier K⁺ channels by entering deeply into a long, narrow pore, displacing K⁺ ions to the outside of the membrane, with this displacement of K⁺ ions contributing to “extra” charge movement. All monoamines are proposed to lie with the “head” amine at a fixed position in the pore, determined by electrostatic interaction, so that zδ is independent of monoamine alkyl chain length. The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases, thus displacing more K⁺ ions to the outside, resulting in charge movement (zδ) increasing with the increase in alkyl chain length.     

Substrate specificity of the Arabidopsis thaliana sucrose transporter AtSUC2

The Arabidopsis sucrose transporter AtSUC2 is expressed in the companion cells of the phloem (specialized vascular tissue) and is essential for the long distance transport of carbohydrates within the plant. A variety of glucosides are known to inhibit sucrose uptake into yeast expressing AtSUC2; however, it remains unknown whether glucosides other than sucrose could serve as transported substrates. By expression of AtSUC2 in Xenopus oocytes and two-electrode voltage clamping, we have tested the ability of AtSUC2 to transport a range of physiological and synthetic glucosides. Sucrose induced inward currents with a K0.5 of 1.44 mM at pH 5 and a membrane potential of -137 mV. Of the 24 additional sugars tested, 8 glucosides induced large inward currents allowing kinetic analysis. These glucosides were maltose, arbutin (hydroquinone-beta-D-glucoside), salicin (2-(hydroxymethyl)phenyl-beta-D-glucoside), alpha-phenylglucoside, beta-phenylglucoside, alpha-paranitrophenylglucoside, beta-paranitrophenylglucoside, and paranitrophenyl-beta-thioglucoside. In addition, turanose and alpha-methylglucoside induced small but significant inward currents indicating that they were transported by At-SUC2. The results indicate that AtSUC2 is not highly selective for alpha-over beta-glucosides and may function in transporting glucosides besides sucrose into the phloem, and the results provide insight into the structural requirements for transport by AtSUC2.     

Stimulation of the glucose carrier SGLT1 by JAK2

JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the ^V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Tumor cell survival depends on availability of glucose. Concentrative cellular glucose uptake is accomplished by Na⁺ coupled glucose transport through SGLT1 (SLC5A1), which may operate against a chemical glucose gradient and may thus be effective even at low extracellular glucose concentrations. The present study thus explored whether JAK2 activates SGLT1. To this end, SGLT1 was expressed in Xenopus oocytes with or without wild type JAK2, ^V617FJAK2 or inactive ^K882EJAK2 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of glucose to the extracellular bath generated a current (I_g), which was significantly increased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Kinetic analysis revealed that coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The stimulating effect of JAK2 expression was abrogated by preincubation with the JAK2 inhibitor AG490. Chemiluminescence analysis revealed that JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I_g during inhibition of carrier insertion by brefeldin A was similar in the absence and presence of JAK2. Thus, JAK2 fosters insertion rather than inhibiting retrieval of carrier protein into the cell membrane. In conclusion, JAK2 upregulates SGLT1 activity which may play a role in the effect of JAK2 during ischemia and malignancy.     

Regulation of Glucose Transporter Expression in Human Intestinal Caco-2 Cells following Exposure to an Anthocyanin-Rich Berry Extract

Polyphenols contained within plant tissues are consumed in significant amounts in the human diet and are known to influence a number of biological processes. This study investigated the effects of an anthocyanin-rich berry-extract on glucose uptake by human intestinal Caco-2 cells. Acute exposure (15 min) to berry extract (0.125%, w/v) significantly decreased both sodium-dependent (Total uptake) and sodium-independent (facilitated uptake) ³H-D-glucose uptake. In longer-term studies, SGLT1 mRNA and GLUT2 mRNA expression were reduced significantly. Polyphenols are known to interact directly with glucose transporters to regulate the rate of glucose absorption. Our in vitro data support this mechanism and also suggest that berry flavonoids may modulate post-prandial glycaemia by decreasing glucose transporter expression. Further studies are warranted to investigate the longer term effects of berry flavonoids on the management of glycaemia in human volunteers.     

Rapid and direct stimulation of hepatic gluconeogenesis by L-triiodothyronine (T3) in the isolated-perfused rat liver

T3 injection into the portal vein of the isolated hypo- and euthyroid liver rapidly stimulated alanine and ¹⁴C-α-amino-isobutyric acid uptake, O₂-consumption, glucose and urea production, and increased the overall, cytoplasmic and mitochondrial energy state. The concentration of the effector molecules long chain acyl CoA, acetyl-CoA, citrat and AMP remained unchanged. After T3 application no alteration in hepatic cAMP content, protein kinase activation, and in the tissue levels of the intermediates of the Embden-Meyerhof-pathway were observed. Our results indicate that T3 rapidly stimulates hepatic glucose production independently of cAMP by increasing amino acid uptake and mitochondrial ATP regeneration and translocation in the cytoplasmic compartment, thus providing both the precursor and energy for gluconeogenesis.     

UPTAKE OF ORGANIC SUBSTRATES BY CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE): EFFECTS OF IONS,IONOPHORES AND METABOLIC AND TRANSPORT INHIBITORS1,2

The uptake of glucose and amino acids by the euryhaline diatom Cyclotella cryptica Reimann, Lewin & Guillard does not appear to be related to proton gradients. Instead, the transport systems for these organic solutes show a strong requirement for the presence of NaCl. The relationship between uptake and NaCl concentration is hyperbolic, with optimal uptake rates being approached at 100 mM NaCl. High concentrations of KCl cause strong reductions in uptake rates. The (Na⁺, K⁺)-stimulated ATPase inhibitor ouabain has no effect on glucose uptake, whereas the diphenolic glucoside phlorizin and its aglucone phloretin are strongly inhibitory. The proton translocating uncoupler CCCP (carbonylcyanide m-chlorophenyl hydrazone) and the ATPase inhibitor DCCD (dicyclohexylcarbodiimide) both almost completely abolish glucose transport, and low concentrations of the ionophares monensin and valenomycin strongly inhibit glucose uptake by the diatom. The requirement of high external NaCl concentrations for glucose transport, and the inhibitory effect an transport of the Na⁺-specific ionophore monensin are consistent with a coupling of Na⁺ and organic substrate transport, but could also be explained by a Na⁺ requirement for glucose binding to a transport carrier, and/or a possible interference with energy producing reactions associated with a monensin-induced collapse of the normal Na⁺ gradient.     

Alkylguanidines as inhibitors of k transport in isolated barley roots

It has been shown that plants can accumulate K⁺ through an energy-dependent process. The effect of alkylguanidines, in particular octylguanidine on the uptake of ⁸⁶Rb⁺ by excised barley roots (Hordeum vulgare var. Apizaco LV-72), has been studied. ⁸⁶Rb⁺ was used as tracer of K⁺. The uptake of ⁸⁶Rb⁺ which is linear with time and shows saturation kinetics is inhibited by octylguanidine. Half-maximal inhibition of ⁸⁶Rb⁺ uptake is attained at 50 μM octylguanidine. Octylguanidine induces a decrease in the V_max of the process and increases the Km of the system for Rb⁺. When the effects of various alkylguanidines were studied, the following order of effectiveness was encountered; octylguanidine = hexilguanidine > butylguanidine > ethylguanidine > guanidine. This suggests that guanidines inhibit Rb⁺ uptake by interacting through its positively charged guanidinium group with a Rb⁺ carrier while the alkyl chain interacts with the hydrophobic milieu of the membrane.     

Catabolism of flavonol glucosides in plant cell suspension cultures

Catabolism of flavonol glucosides was investigated in plant cell suspension cultures using kaempferol 3-O-β-d-glucoside and kaempferol 7-O-β-d-glucoside labelled with ¹⁴C either in the glucose or in the flavonol moiety. Catabolic rates of glucosides were compared with those of free glucose and kaempferol. All substrates were degraded efficiently by cell cultures of mungbean, soybean, garbanzo bean and parsley. Based on ¹⁴CO₂-formation, glucose from position 3 of kaempferol is 3–5 times more rapidly metabolized than that from position 7. The flavonol nucleus from both isomers is, however, oxidized to the same extent with a considerable portion of the flavonol being incorporated into insoluble polymeric cell material.