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Review Sulfate Safety and Quality

Nicola Volpi

Department of Life Sciences, Laboratory of Biochemistry and Glycobiology, University of Modena and Reggio Emilia, 41125 Modena, Italy; [email protected]; Tel.: +39-59-2055543; Fax: +39-59-2055548

 Academic Editor: Cédric Delattre  Received: 7 March 2019; Accepted: 9 April 2019; Published: 12 April 2019

Abstract: The industrial production of (CS) uses animal tissue sources as raw material derived from different terrestrial or marine species of animals. CS possesses a heterogeneous structure and physical-chemical profile in different species and tissues, responsible for the various and more specialized functions of these macromolecules. Moreover, mixes of different animal tissues and sources are possible, producing a CS final product having varied characteristics and not well identified profile, influencing oral absorption and activity. Finally, different extraction and purification processes may introduce further modifications of the CS structural characteristics and properties and may lead to extracts having a variable grade of purity, limited biological effects, presence of contaminants causing problems of safety and reproducibility along with not surely identified origin. These aspects pose a serious problem for the final consumers of the pharmaceutical or products mainly related to the traceability of CS and to the declaration of the real origin of the active ingredient and its content. In this review, specific, sensitive and validated analytical quality controls such as electrophoresis, eHPLC (enzymatic HPLC) and HPSEC (high-performance size-exclusion chromatography) able to assure CS quality and origin are illustrated and discussed.

Keywords: chondroitin sulfate; ; ; ; food supplements

1. Chondroitin Sulfate Structure (HA), (KS), chondroitin sulfate (CS)/ (DS) and (HS)/ are natural macromolecules and the main complex heteropolysaccharides belonging to the class of glycosaminoglycans (GAGs) [1,2]. Apart from KS, which is composed of and N-acetyl-d-galactosamine (GalNAc) [3], the backbone of the other GAGs is commonly represented by typical sequences constituted of alternating units of uronic acids, glucuronic (GlcA) or iduronic (IdoA), and amino sugars, N-acetyl-d- (GlcNAc) or GalNAc [4]. KS, CS/DS and HS/heparin are sulfated macromolecules possessing different degrees of charge density thanks to the presence of sulfate groups in varying amounts and located in different positions. Moreover, besides the variability in sulfate groups, these are very heterogeneous in terms of molecular mass, physicochemical properties, and biological and pharmacological properties [1–4]. CS with different and overall carbohydrate backbones may be biosynthesized possessing various number and position of sulfate groups [4] (Figure1). Consequently, CS is a largely heterogeneous GAG formed of alternate and variously sulfated disaccharides of GlcA and GalNAc linked by β(1 3) bonds. Moreover, the different disaccharides are linked to each other by β(1 4) → → bonds. CSA and CSC are constituted by disaccharides sulfated in position C4 or C6 of the GalNAc residue, respectively, and minor percentages of the monosulfated disaccharide on C2 of GlcA are possible. Besides the main presence of these two kinds of disaccharide units monosulfated in position C4 or C6 of GalNAc, disaccharides with a different number and position of sulfate groups can be present, in various percentages, within the carbohydrate chains [4–6]. Various kinds of disulfated disaccharides may be present in the CS structure in various amounts also in relation to specific animal

Molecules 2019, 24, 1447; doi:10.3390/molecules24081447 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 13

C4 or C6 of GalNAc, disaccharides with a different number and position of sulfate groups can be Molecules 2019, 24, 1447 2 of 13 present, in various percentages, within the carbohydrate chains [4–6]. Various kinds of disulfated disaccharides may be present in the CS structure in various amounts also in relation to specific animal sourcessources (as (as in in cartilaginous cartilaginous or or bony bony fishes, fishes, seesee below),below), such as as the the disaccharide disaccharide disulfated disulfated in inposition position C2C2 of GlcAof GlcA and and C6 C6 of GalNAcof GalNAc (the (the so-called so-called disaccharide disaccharide D), D), in in position position C4 C4 and and C6 C6 of of the the GalNAc GalNAc unit (theunit disaccharide (the disaccharide E) or C2 E) ofor GlcAC2 of GlcA and C4 and of C4 GalNAc of GalNAc (the (the disaccharide disaccharide B) (FigureB) (Figure1). 1). Finally, Finally, a lowa low percentage of the non-sulfated disaccharide is almost always present in the CS backbone while percentage of the non-sulfated disaccharide is almost always present in the CS backbone while the the fully tri-sulfated disaccharide is generally in trace amount [4–6]. fully tri-sulfated disaccharide is generally in trace amount [4–6].

Figure 1. Structure of the disaccharides present in chondroitin sulfate backbone constituted of d-glucuronicFigure 1. Structure acid and ofN -acetyl-the disaccharidesd-galactosamine present linked in chondroitin by β(1 sulfate3) bonds. backbone The various constituted disaccharides of D- → areglucuronic linked each acid other and by N-acetyl-β(1 4)D-galactosamine linkages. Minor linked percentages by β(1→ of3) verybonds. rare The disaccharides various disaccharides may also are have → a sulfatelinked group each other in position by β(1→ C34) oflinkages. the glucuronic Minor percentages acid. Ac, acetylof very group. rare disaccharides may also have a sulfate group in position C3 of the . Ac, acetyl group. Besides the above-illustrated CS structures, a peculiar CS known as CSB or DS is knownBesides in nature, the formed above-illustrated of disaccharide CS structures, units of a IdoApeculiar and CS GalNAc polysaccharide mainly sulfatedknown as in CSB position or DS C4 withis aknown minor in and nature, variable formed content of disaccharide of the disulfated units of IdoA disaccharide and GalNAc in position mainly C4sulfated of GalNAc in position and C4 C2 of IdoUAwith [a4 ].minor However, and variable the biosynthetic content of the processes disulfated generally disaccharide produce in position highly C4 modified of GalNAc oligosaccharide and C2 of IdoUA [4]. However, the biosynthetic processes generally produce highly modified oligosaccharide domains separated by regions having a low-degree of structural modifications introducing further domains separated by regions having a low-degree of structural modifications introducing further heterogeneity. Consequently, the CS and DS chains may be hybrid polymers of low modified (CS) and heterogeneity. Consequently, the CS and DS chains may be hybrid polymers of low modified (CS) highly modified (DS) domains [4]. The heterogeneity introduces a great variability in the and highly modified (DS) domains [4]. The sulfation heterogeneity introduces a great variability in positionthe position of sulfate of groups sulfate and groups in CS charge and in density. CS charge Moreover, density. the Moreover, different number the different of disaccharide number units of formingdisaccharide the CS units polymer forming and the CS overall polymer molecular and the weight overall generatedmolecular weight are other generated key factors are other influencing key its properties.factors influencing Finally, its theproperties. combination Finally, of the the combination different sulfated of the different disaccharides sulfated anddisaccharides oligosaccharide and domainsoligosaccharide produces domains a huge produces number ofa huge heterogeneous number of heterogeneous CS polymers CS possessing polymers possessing various, specific various, and specializedspecific and biological specialized and biological pharmacological and pharmacological functions [1, 2functions]. [1,2].

2. Chondroitin2. Chondroitin Sulfate Sulfate Activity Activity CSCS is ais natural a natural biomacromolecule biomacromolecule abundantly abundantly distributed in in virtually virtually all all invertebrates invertebrates and and vertebratesvertebrates (and (and consequently consequently in in humans) humans) and and involved involved in in many many biological biological processes processes [ 1[1,5,7,8].,5,7,8]. BasedBased on its organism-to-organismon its organism-to-organism and tissue-to-tissue and tissue-to-tissue structural structural diversity diversity in chain in lengthchain length and sulfation and sulfation patterns, CSpatterns, provides CS specific provides biological specific functionsbiological atfunctions molecular, at molecular, cellular and cellular organ and level organ such level as such cell adhesion,as cell celladhesion, division and cell di divisionfferentiation, and differentiation, morphogenesis, morphogenesis, organogenesis organogenesis and neural network and neural formation network [9–11 ]. Moreover,formation CS [9–11]. is particularly Moreover, abundant CS is particularlyin all mammalian abundant connective in all mammalian tissues, especially connective in the tissues, , skin,especially blood vessels, in the ligamentscartilage, skin, and tendonsblood vessels, with di ligamentsfferent structure and and level with of sulfationdifferent structure [12]. In addition and to itslevel conventional of sulfation structural [12]. In roles addition in the to composition its conventional of extracellular structural matrix roles in and the formation composition of organs of and formation of organs and tissues, such as and , recent and tissues, such as cartilages and bones, recent evidence demonstrates that CS chains are involved in evidence demonstrates that CS chains are involved in important biological functions in inflammation, important biological functions in inflammation, infection and wound repair [7–11]. infection and wound repair [7–11]. The biological effects of CS are related to its ability to interact with a wide variety of other bio(macro)molecules including (but not limited to) matrix molecules, growth factors, inhibitors, cytokines, chemokines, adhesion molecules and pathogen virulence factors via aspecific/specific sulfated saccharide domains within the chains [11–13]. In fact, along with aspecific interactions due to its numerous negative charges provided by the sulfate and carboxyl groups, CS is involved in specific Molecules 2019, 24, 1447 3 of 13 binding to bioactive molecules for the presence of peculiar functional domain structures which are formed by combinations of the various disaccharide units [11–13] (see Figure1). Osteoarthritis (OA) is a global issue affecting all known countries and ethnicities with huge incidence and economic impact [14]. OA particularly affects the older population producing variable degrees of limitation of movement and major activities of daily living. Moreover, OA is expected to dramatically rise in the future, in particular for the population above 70 years of age [14–16], also related to the observed increase of obesity in western populations [17,18]. Current treatments are mainly based on pain reduction [19] and treatments having the aim to reduce the progression of the disease, such as the S/DMOADs (structure/disease modifying anti-osteoarthritis drugs) [20,21]. CS inhibits the extracellular involved in the of connective tissues, and it is able to stimulate production by chondrocytes as well as reduce the cartilage cytokine production but also to induce apoptosis of articular chondrocytes [8,22]. Furthermore, CS increases the intrinsic viscosity of the synovial liquid and, in bones, it accelerates the mineralization process and repair [8,22]. Overall, CS has a key role in articular and metabolism by controlling cartilaginous matrix integrity and bone mineralization. Consequently, with others, treatment with CS is reported to improve OA symptoms and to reduce the disease. In fact, in many published studies, CS has demonstrated to be a symptomatic slow acting drug for the treatment of OA (SYSADOA) and to have disease modifying properties in long-term treatments (so-called DMOAD) (see [20–24] for reviews).

3. Safety and Quality Concerns of Chondroitin Sulfate CS, according to its nature, must be extracted from animal sources and submitted to purification processes for commercial purposes [5,6]. Actual commercial production of CS is based on terrestrial raw material, from bovine, porcine, and chicken, or marine sources such as cartilaginous fish, sharks and skate, but also bony fishes ([5,6,25] and personal information). Moreover, a mix of all these sources is possible, producing a CS final product with mixed characteristics and properties. The use of possible not controlled raw animal material (tissues, bones and cartilages but also soft organs) poses the problem of a final product with not controlled structure and poor reproducibility along with the not well identified origin with the consequence of variable grade of purity, biological effects, presence of contaminants, clinical efficacy and safety [5,6,26]. Besides the source material, manufacturing processes, the presence of contaminants and other natural biomolecules, as well as many other factors, contribute to the quality, structure and properties of the final product influencing CS overall biological, nutraceutical and pharmacological capacities. In fact, as well known, CS possesses a complex structure strictly depending on the tissue, organ and species but also on the age of the animals [5,6]. Moreover, the biological and nutraceutical properties may vary with the structure as well as the oral absorption that is influenced by the different CS physicochemical properties [27–29]. Furthermore, due to the extractive origin of CS and to the different purification processes applied, the presence of bacteria, virus or prions cannot be excluded [30]. Additionally, other various natural bioactive (macro)molecules may be present in different content as contaminants in CS final extracts [5,6], and voluntary adulteration by artificial compounds is also known. Finally, a restriction use related to religious issues is possible (see below for these CS concerns). Overall, depending on the source origin and on the manufacturing processes, variable CS final products for structure, quality and activity may be produced (Table1)[5,6,26]. Molecules 2019, 24, 1447 4 of 13

Table 1. Main general characteristics and properties of animal CS for commercial purposes.

Variable and generally not defined and heterogeneous source of extraction Possible cross-contamination between sources of different origin Possible presence of bacteria, virus and/or prions High content of proteins that are not characterized (up to 5–10%). Some of these proteins may have allergenic potential able to develop immune reactions Variable content of immunogenic keratan sulfate and other natural biopolymers Variable purity Heterogeneous structure and physicochemical properties. Variable molecular mass, polydispersity and charge density Process of extraction generally not controlled causing possible modifications of the CS structure, such as desulfation and/or depolymerization Possible intentional adulteration by artificial (macro)molecules Possible batch-to-batch variability In general, no evaluation of any biological activity

CS from various sources has a carbohydrate backbone formed of disaccharides with sulfate groups in different percentages and positions (see Figure1) producing polymers possessing di fferent charge densities. Furthermore, due to the biosynthetic processes, CS macromolecules with different grades of polymerization may be generated with various molecular masses and polydispersity related to specific tissues, organs and species. Because of these structural variations, and the further possible presence of specific oligosaccharide sequences (and purity, see below) of the preparations for therapy applications or in nutraceuticals, CS may have different biological properties and capacities [5,26]. According to this structural variability, different and peculiar activities have been reported depending on the CS structure [5]. Finally, bioavailability and pharmacokinetic have been reported to change depending on CS structural properties and origin when orally administered during therapy [27–29]. Extraction and purification processes may further produce CS preparations having different structures introducing modifications of the macromolecular characteristics and chemical properties by controlled as well as undesired depolymerization, desulfation and/or chemical modifications [6]. Another key point is related to the accurate evaluation of the CS content and purity when the preparation is produced for therapeutic or nutraceutical applications. In fact, the CS content and purity may change according to the manufacturing processes and/or tissue sources. Depending on the purification protocols, the extracts may have a variable content and purity of CS due to the presence of polluting inorganic or organic side-products (Table1). All the above-mentioned aspects pose a serious problem for the pharmaceutical or nutraceutical Companies having the necessity to declare the real origin of the active ingredients and the label traceability of CS from the raw material up to the finished products. Moreover, an accurate label indication of the CS content is of paramount importance to assure to the final consumer a real dose administration also considering the possible intentional adulteration by potentially dangerous (macro)molecules (see below) mimicking CS structure and properties.

3.1. Natural Contaminations As already mentioned, commercial CS produced from animal sources requires long and complex procedures of extraction and purification with the aim to eliminate or reduce the content of the other (macro)molecules present as natural contaminants [26] (Figure2). At the same time, contamination of CS may be accidentally caused by the same extraction and purification processes if not well controlled. Obviously, as more steps of purification are adopted having differential capacities in the elimination of natural contaminants of various nature, CS final products with higher quality may be obtained. On the Molecules 2019, 24, 1447 5 of 13 other hand, the production of high-quality CS of pharmaceutical grade generally requires high costs of productionMoleculesMolecules 20192019 and,, 2424 quality,, xx FORFOR PEERPEER analytical REVIEWREVIEW controls. 5 5 ofof 1313

FigureFigureFigure 2. Natural 2. 2. NaturalNatural contaminations contaminations contaminations eventually eventually eventually present present present in chondroitin in in chondroitin chondroitin sulfate preparations sulfate sulfate preparations preparations and responsible and and forresponsibleresponsible reduced title forfor and reducedreduced possible titletitle side-eandand possiblepossibleffects. side-effects.side-effects. 3.1.1. Polysaccharides 3.1.1.3.1.1. PolysaccharidesPolysaccharides DependingDependingDepending on on onthe the theanimal animal animal source source source and onand and the on on extraction the the extraction extraction and purification and and purification purification procedures procedures procedures that generally that that aregenerallygenerally complex areare and complexcomplex require andand long requirerequire time, longlong CS extracts time,time, CSCS and extractsextracts final andand products finalfinal productsproducts for commercial forfor commercialcommercial purposes purposespurposes are also pollutedareare alsoalso with pollutedpolluted variable withwith percentages variablevariable percentagespercentages of other polysaccharides ofof otherother polysaccharidespolysaccharides that are co-extractedthatthat areare co-extractedco-extracted with CS withwith during CSCS its preparationduringduring itsits (Figure preparationpreparation3). (Figure(Figure 3).3).

Figure 3. Natural polysaccharides eventually present in chondroitin sulfate preparations and the FigureFigure 3. 3. NaturalNatural polysaccharides polysaccharides eventuallyeventually present present inin chondroitinchondroitin sulfate sulfate preparationspreparations andand the the consequences on the chondroitin quality and title. consequencesconsequences onon thethe chondroitinchondroitin qualityquality andand title.title. HA in particular, and DS with its carbohydrate backbone made of , have been HAHA inin particular,particular, andand DSDS withwith itsits carbohydratecarbohydrate backbonebackbone mademade ofof iduroniciduronic acid,acid, havehave beenbeen detected in raw materials and formulations and reported in scientific papers [31] due to their ubiquitous detecteddetected in in raw raw materials materials and and formulations formulations and and reported reported in in scientific scientific papers papers [31] [31] due due to to their their presenceubiquitousubiquitous in tissues presencepresence from inin tissuestissues which fromfrom CS is whichwhich generally CSCS isis produced. generallygenerally produced.produced. Moreover, Moreover,Moreover, KS has beenKSKS hashas detected beenbeen detecteddetected in many batchesinin manymany of batches CSbatches produced ofof CSCS producedproduced from shark fromfrom cartilage, sharkshark cartilage,cartilage, averaging averagingaveraging 16% 16%16% of the ofof thethe total totaltotal GAGs GAGsGAGs [ 32 [32,33][32,33],33] (Figure (Figure(Figure 4 ). Indeed,4).4). Indeed,Indeed, as KS asas is KSKS involved isis involvedinvolved in the inin structure thethe structurestructure of the ofof the proteoglycanthe proteoglycanproteoglycan aggrecanaggrecan located locatedlocated in inin all allall cartilages, cartilages,cartilages, it itit has beenhashas detected beenbeen detecteddetected in extracts inin extractsextracts and finished andand finishedfinished preparations preparationspreparations of various ofof variousvarious origin origin alongorigin withalongalong CS withwith extracted CSCS extractedextracted from the samefromfrom cartilages thethe samesame [34 cartilagescartilages]. A total [34].[34]. of 15 AA samples, total total ofof 1515 including samples,samples, four includingincluding samples fourfour of samples CSsamples as laboratory ofof CSCS asas reagents,laboratorylaboratory one samplereagents,reagents, of CS oneone as asamplesample food additive ofof CSCS asas a anda foodfood ten additiveadditive samples andand of ten dietaryten samplessamples supplements ofof dietarydietary containingsupplementssupplements CS, containingcontaining were examined CS,CS, forwerewere the presence examined examined of forKS for by the the using presence presence specific of of KS KS immunodi by by using usingff usion specific specific and immunodiffusion immunodiffusion enzyme-linked immunosorbent and and enzyme-linked enzyme-linked assay (ELISA).immunosorbentimmunosorbent Apart from assay assay the three (ELISA). (ELISA). samples Apart Apart of CSfrom from as the thelaboratory three three samples samples reagents, of of all CS CS samples as as laboratory laboratory were found reagents, reagents, to contain all all varyingsamplessamples amounts werewere foundfound of KS toto [34 containcontain]. Finally, varyingvarying it is worth amountsamounts mentioning ofof KSKS [34].[34]. that Finally,Finally, KS possesses itit isis worthworth immunogenic mentioningmentioning thatthat capacities KSKS possesses immunogenic capacities able to develop immune reactions [35]. Depending on the animal ablepossesses to develop immunogenic immune reactions capacities [35 able]. Depending to develop onimmune the animal reactions raw [35]. material Depending used for on CSthe extraction,animal raw material used for CS extraction, HS may also be present due to its comparable physicochemical HSraw may material also be presentused for dueCS extraction, to its comparable HS may physicochemicalalso be present due properties to its comparable with CS, physicochemical in particular for its properties with CS, in particular for its nature of sulfated heteropolysaccharide [12]. natureproperties of sulfated with heteropolysaccharideCS, in particular for its [nature12]. of sulfated heteropolysaccharide [12].

Molecules 2019, 24, 1447 6 of 13 Molecules 2019, 24, x FOR PEER REVIEW 6 of 13 Molecules 2019, 24, x FOR PEER REVIEW 6 of 13

FigureFigure 4. Acetate4. Acetate cellulose cellulose electrophoresis electrophoresis ableable toto detect the presence presence of of (A (A) )keratan keratan sulfate sulfate (KS) (KS) or or (B)Figure dermatan(B) dermatan 4. Acetate sulfate sulfate cellulose (DS) (DS) and electrophoresis/or and/or heparan heparan sulfate able sulfate (HS)to detect in (HS) chondroitin the in presence chondroitin sulfate of ( A sulfate (CS)) keratan preparations (CS) sulfate preparations (KS) (material or owned((materialB) dermatan by the owned same sulfate by author). the (DS) same and/or author). heparan sulfate (HS) in chondroitin sulfate (CS) preparations (material owned by the same author). TheThe presence presence of of these these natural natural polymers polymers inin CSCS products,products, also also in in high high percentages, percentages, alerts alerts the the manufacturersmanufacturersThe presence for for improved of improved these isolationnatural isolation polymers procedures procedures in asCS well asproducts, well as the as supervisoryalso the supervisoryin high agenciespercentages, agencies for betteralerts for better audits.the In addition,manufacturersaudits. In thisaddition, finding for improvedthis also finding compromises isolation also compromises procedures the desired the as desired amounts well asamounts the of the supervisory of real the ingredient real ingredient agencies specified for specified better on the labelaudits.on claims the Inlabel andaddition, claims forewarns this and finding forewarns the pharmacopeias also thecompromises pharmacopeias to update the desired theirto update monographs.amounts their of monographs. the In real fact, ingredient general In fact, non-specificspecified general analyticalonnon-specific the label methods claimsanalytical such and methods as forewarns CPC (cetylpyridiniumsuch the as pharmacopeias CPC (cetylpyridinium chloride) to update titration chloride) their andmonographs. titration carbazole and Incarbazole assay fact, aregeneral assay unable to detectnon-specificare unable the presenceto analytical detect ofthe methods other presence polysaccharides such of otheras CPC polysaccharides (cetylpyridinium in CS products in CSchloride) due products to titration their due quite to and their similarcarbazole quite properties similar assay andareproperties chemical unable to and structures detect chemical the (seepresence structures below of for other (see further belowpolysaccharides analytical for further in considerations). analyticalCS products considerations). due Finally, to their thequite Finally, presence similar the of properties and chemical structures (see below for further analytical considerations). Finally, the variablepresence amounts of variable of the amounts immunogenic of the immunogenic KS poses serious KS poses problems serious related problems to the related possible to the development possible presencedevelopment of variable of immunogenic amounts of adverse the immunogenic reactions (Figure KS poses 3). serious problems related to the possible of immunogenic adverse reactions (Figure3). development of immunogenic adverse reactions (Figure 3). 3.1.2.3.1.2. Other Other Biomolecules Biomolecules 3.1.2. Other Biomolecules Other (macro)molecules possessing similar properties of CS are copurified during CS extraction Other (macro)molecules possessing similar properties of CS are copurified during CS extraction fromOther tissues, (macro)molecules such as nucleic acidspossessing [31] and similar proteins properties (Figure of 5). CS In are fact, copurified animal tissues during and CS organsextraction are from tissues, such as nucleic acids [31] and proteins (Figure5). In fact, animal tissues and organs fromrich in tissues, DNA, suchRNA as and nucleic proteins, acids and [31] variable and proteins content (Figure of these 5). biomolecules In fact, animal are tissues present and in CSorgans extracts are are rich in DNA, RNA and proteins, and variable content of these biomolecules are present in CS richdepending in DNA, on RNA the source and proteins, and on andthe purification variable content protocols of these adopted. biomolecules Moreover, are somepresent of thesein CS proteinsextracts extractsdependingmay dependinghave allergenicon the onsource theand/or sourceand onintolerance andthe purification on the capacity purification protocols able to protocols adopted.develop immuneMoreover, adopted. reactions. Moreover,some of theseFinally, some proteins ofthese these proteinsmaymacromolecules have may allergenic have allergenicalso and/orgive positive andintolerance/or intoleranceresponse capacity to capacityaspecific able to develop ableanalytical to develop immune controls immune reactions. such as reactions. CPCFinally, assay these Finally, [6] thesemacromolecules(Figure macromolecules 5). also also give give positive positive response response to aspecific to aspecific analytical analytical controls controls such such as CPC as CPC assay assay [6] [6] (Figure(Figure5). 5).

Figure 5. Natural biomolecules possibly present in chondroitin sulfate preparations and their related FigureFigureproblems. 5. 5. NaturalNatural biomolecules biomolecules possibly possibly present present in chondroitin in chondroitin sulfate sulfatepreparations preparations and their andrelated their relatedproblems. problems.

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Molecules 2019, 24, x FOR PEER REVIEW 7 of 13 3.1.3. Infective Agents 3.1.3. Infective Agents The animal origin of CS may pose safety problems due to the possible presence of pathogen contaminationThe animal and origin transmissible of CS may infective pose safety agents problems such due as bacteriato the possible residues, presence viruses of pathogen and prions potentiallycontamination causing and spongiform transmissible encephalopathy infective agents in bovines such as (BSE), bacteria foot-and-mouth residues, viruses disease, and influenza prions spreadpotentially in birds causing and other spongiform animal encephalopathy diseases (Figure in bovines5). The (BSE), possible foot-and-mouth presence of disease, infective influenza agents in spread in birds and other animal diseases (Figure 5). The possible presence of infective agents in the the organs and tissues used for CS production requires the application of specific chemical steps organs and tissues used for CS production requires the application of specific chemical steps able to able to destroy or inactivate and eliminate bacteria, viruses and prions [36,37] possibly introducing destroy or inactivate and eliminate bacteria, viruses and prions [36,37] possibly introducing chemical chemical modifications in the CS structure such as degradation, desulfation, oxidation of carbohydrate modifications in the CS structure such as degradation, desulfation, oxidation of carbohydrate chains, chains,introduction introduction of chemical of chemical groups, etc. groups, In fact, etc. prions In fact,adhere prions very tenaciously adhere very to surfaces, tenaciously making to surfaces, them makinghard them to remove, hard to and remove, their and resistance their resistance to protease to protease treatment, treatment, certain chemical certain chemical agents and agents heat and heatdenaturation denaturation represents represents a majora major concern concern for for pharmaceutical pharmaceutical products products derived derived from from terrestrial terrestrial animalsanimals [36 [36].]. Moreover, Moreover, the the purification purification steps steps usefuluseful to remove remove the the infective infective agents agents can can also also affect affect the the biologicalbiological capacities capacities of of extracted extracted CS CS (Figure (Figure6 ).6).

FigureFigure 6. Common6. Common possible possible pathogen pathogen contaminationcontamination and transmissible transmissible infective infective agents agents in inchondroitin chondroitin sulfatesulfate preparations preparations and and their their e ffeffects.ects.

AnotherAnother key key point point is is the the adoption adoption of of specificspecific and selective selective analytical analytical assays assays to todetect detect the the possible possible presencepresence of of the the di differentfferent infective infective agentsagents in in the the final final products. products. In fact, In fact, the thepresence presence of prions of prions may be may be easily demonstrated with with a a diagnostic diagnostic test. test. However, However, the the current current available available analytical analytical assays assays are are characterizedcharacterized by by high high costs costs and and limited limited toto specializedspecialized laboratories laboratories [38]. [38]. Finally, Finally, other other pathogens pathogens show show similarsimilar attributes attributes as as prions prions for for the the potential potential riskrisk ofof carry-overcarry-over into into final final purified purified products. products.

3.2.3.2. Intentional Intentional Adulterations Adulterations BesidesBesides the the presence presence of of natural natural contamination, contamination, finished finished CS CS preparations preparations may may also also be beadulterated adulterated by theby voluntarythe voluntary addition addition of cheaper of cheaper polysaccharides polysaccharides and organic and organic derivatives derivatives unable to unable be determined to be withdetermined usual analytical with usual controls analytical utilized controls in theutilized nutraceutical in the nutraceutical industry industry (Figure 7(Figure). In fact, 7). In CS fact, is CS well is well known to be one of the most adulterated supplements in the market, a widespread and known to be one of the most adulterated supplements in the market, a widespread and probably probably underestimated problem, abetted by the lack of industry-wide specific and sensitive quality underestimated problem, abetted by the lack of industry-wide specific and sensitive quality control control analytical methods with several of the adulterated agents going undetected [6,39]. analytical methods with several of the adulterated agents going undetected [6,39].

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FigureFigure 7. Artificially7. Artificially adulterated adulterated chondroitin chondroitin sulfatesulfate by identified identified substances substances and and their their consequences. consequences.

ApproximatelyApproximately 80–90% 80–90% of of the the CS CS supplied supplied toto the US and and Europe Europe markets markets is isproduced produced in inChina China andand intentional intentional adulteration adulteration is possiblyis possibly practiced practiced by by nutraceutical nutraceutical manufacturers manufacturers globally globally with with the the aim to reduceaim to reduce ingredient ingredient costs bycosts using by using cheaper cheaper substances substances [40]. [40]. The The price price pressure pressure is is also also responsible responsible for poorfor purification poor purification procedures procedures with thewith production the production of CS of preparationsCS preparations rich rich in saltsin salts and and having having low low title buttitle also but to minimizealso to minimize analytical analytical controls controls to only to common only common non-specific non-specific and inexpensive and inexpensive tests tests [40]. [40]. In this scenario,In this somescenario, raw some material raw andmaterial dietary and supplementdietary supplement products products contain contain less than less the than claimed the claimed amount of CS,amount in some of CS, cases in some as little cases as 5–10%as little [41 as ,42 5–10%] with [41,42] a huge with fraud a huge to clients, fraud to traders clients, and traders consumers and (Figureconsumers7). In fact, (Figure many 7). studiesIn fact, availablemany studies in scientific available literature in scientific demonstrate literature demonstrate the poor quality the poor and a titlequality not conforming and a title to not label conforming declaration to of label many declaration CS finished of many products CS finished in several products countries in [ several6,41–45 ]. countriesThe application [6,41–45]. of specific analytical methods such as eHPLC (enzymatic HPLC), HPSEC (high-performanceThe application size-exclusion of specific analytical chromatography) methods such and as electrophoresis eHPLC (enzymatic (Figure HPLC),4 and HPSEC see below (high- for performance size-exclusion chromatography) and electrophoresis (Figure 4 and see below for analytical methods) on commercially available nutraceuticals has demonstrated the poor quality of analytical methods) on commercially available nutraceuticals has demonstrated the poor quality of many of these products in the global market showing a lower concentration of CS than specified many of these products in the global market showing a lower concentration of CS than specified on on the label as well as their intentionally adulteration [39,41,42]. The adulteration is obtained with the label as well as their intentionally adulteration [39,41,42]. The adulteration is obtained with molecules that may interfere with aspecific widely-used analytical methods of CS quantitation, such as molecules that may interfere with aspecific widely-used analytical methods of CS quantitation, such theas CPC the titrationCPC titration and carbazoleand carbazole assay, assay, to give to give an artificiallyan artificially inflated inflated estimate estimate of of CS CS concentration concentration [6]. Many[6]. (macro)moleculesMany (macro)molecules have beenhave been identified identified for CS for adulteration, CS adulteration, such such as carrageenan,as carrageenan, proteins proteins and surfactants,and surfactants, cheaper cheaper polysaccharides, polysaccharides, sodium alginate, alginate, propylene propylene glycol alginate alginate sulfate sulfate sodium, sodium, sodiumsodium hexametaphosphate hexametaphosphate commonly commonly known with with the the name name of ofCalgon Calgon and and used used as a asdetergent a detergent or or -treatmentwater-treatment additive additive [39], [39], maltodextrin maltodextrin and and lactose lactose [42] [42 (Figure] (Figure 7).7). These These substances substances can can be be separatedseparated from from real real CS CS by by their their di ffdifferenceerence in in electrophoretic electrophoretic mobility mobility (Figure (Figure4) 4) and and a a correct correct title title of of CS mayCS be may obtained be obtained by other by other specific specific analytical analytical approaches approaches such such as eHPLCas eHPLC and and HPSEC HPSEC (see (see below), below), and notand with not commonly-used with commonly-used methods methods [6,39, 41[6,39,41,42],42] (Figure (Figure7). 7).

4. Analytical4. Analytical Quality Quality Controls Controls CSCS for for pharmaceutical pharmaceutical applications applications is is strictly strictly evaluated for for quality, quality, content, content, structural structural characterizationcharacterization and and parameters parameters (such (such as as chargecharge density and and molecular molecular mass) mass) by by means means of ofsensitive, sensitive, specific,specific, validated validated and publishedand published analytical analytical approaches approaches [6]. These [6]. features These features are of paramount are of paramount importance to ensureimportance therapeutic to ensure reproducibility therapeutic reproducibility and safe use, and andsafe use, to protect and to protect patients patients from inefromffective ineffective and /or and/or unsafe and potentially dangerous drugs. As discussed below, is it possible to affirm the same unsafe and potentially dangerous drugs. As discussed below, is it possible to affirm the same for food for food and nutraceutical CS preparations? and nutraceutical CS preparations? European and USA pharmacopoeias require some analytical tests for CS identification such as European and USA pharmacopoeias require some analytical tests for CS identification such infrared spectroscopy, specific optical rotation, intrinsic viscosity, electrophoresis and CPC titration as infrared spectroscopy, specific optical rotation, intrinsic viscosity, electrophoresis and CPC [46,47]. Some of these assays are very old and usually no longer used in modern quality control titrationlaboratories[46,47] .due Some to oftheir these lack assays of specificity, are very oldlow andsensitivity usually and no longerpoor reproducibility. used in modern In quality particular, control laboratories due to their lack of specificity, low sensitivity and poor reproducibility. In particular, infrared spectroscopy and specific optical rotation cannot give specific information belonging to Molecules 2019, 24, 1447 9 of 13 Molecules 2019, 24, x FOR PEER REVIEW 9 of 13 manyinfrared other spectroscopy (macro)molecules, and specific suchas optical the absorption rotation cannot of chemical give specific groups information in the spectrum belonging of infrared to wavelengthsmany other (for (macro)molecules, infrared spectroscopy) such as the [ 48absorption] or the capacityof chemical to groups rotate thein the plane spectrum of polarization of infrared of plane-polarizedwavelengths (for light infrared by chiral spectroscopy) chemical compounds [48] or the suchcapacity as carbohydrates to rotate the plane (for opticalof polarization rotation) of [49 ]. Intrinsicplane-polarized viscosity measurementlight by chiral ischemical a very compounds old technique such for as the carbohydrates determination (for of optical the molecular rotation) [49]. weight of aIntrinsic polymer viscosity replaced measurement by the more is a modernvery old technique size-exclusion for the chromatography determination of the coupled molecular with weight various detectorsof a polymer [50] (Figure replaced8). by The the industry-wide more modern methodsize-exclusion for analysis chromatography of CS is CPC coupled titration. with However,various severaldetectors flaws [50] in this(Figure old testing8). The methodindustry-wide show relevantmethod for limits, analysis as it canof CS be is tricked CPC titration. by the use However, of natural several flaws in this old testing method show relevant limits, as it can be tricked by the use of natural macromolecules and/or artificial adulterants and cannot distinguish between CS and other material macromolecules and/or artificial adulterants and cannot distinguish between CS and other material having similar physicochemical properties, e.g., nucleic acids, anionic proteins, other polysaccharides, having similar physicochemical properties, e.g., nucleic acids, anionic proteins, other etc. [6]. Moreover, CPC assay is also influenced by the molecular weight of tested CS as well as for polysaccharides, etc. [6]. Moreover, CPC assay is also influenced by the molecular weight of tested the carbazole assay that is another non-specific quantitative test [6]. On the contrary, agarose-gel CS as well as for the carbazole assay that is another non-specific quantitative test [6]. On the contrary, electrophoresisagarose-gel electrophoresis according to European according pharmacopoeia, to European pharmacopoeia, electrophoresis electrophoresis on acetate of on cellulose acetate (ACE) of accordingcellulose to (ACE) US pharmacopoeia, according to US andpharmacopoeia, HPSEC and and eHPLC HPSEC according and eHPLC to the according AOAC to (Association the AOAC of Offi(Associationcial Analytical of Official Chemists) Analytical [51] are Chemists) highly specific [51] are analytical highly specific approaches analytical for the approaches determination for the of CS quality,determination quantity, of chemical CS quality, properties quantity, and chemical structure. properties and structure.

FigureFigure 8. Specific8. Specific analytical analytical procedures procedures able able to distinguish to distinguish between between chondroitin chondroitin sulfate sulfate produced produced from difffromerent different origin and origin toassess and to CSassess quality CS quality and real and quantity. real quantity.

BesidesBesides the the low low title, title, alsoalso origin of of CS CS has has the the been been reported reported to be to a bepuzzle a puzzle and in and general in general not notdeclared declared on on product product labels labels [6]. The [6]. origin The originof the CS of utilized the CS in utilized a finished in product a finished can only product be detected can only be detectedby rather by expensive rather expensive and detailed and detailed biochemical/structural biochemical/structural analyses analyses by bydetermining determining the the disaccharidedisaccharide/oligosaccharide/oligosaccharide pattern pattern and and molecular molecular mass mass parameters parameters [6]. [6 ]. According According to to this, this, a a combinationcombination of specificof specific analytical analytical methods methods to define to define the finalthe final quality, quality, origin origin and propertiesand properties of the of product the is required.product is Such required. methods Such include methods electrophoresis, include electrophoresis, HPSEC, selective HPSEC, enzymaticselective enzymatic treatment treatment and further separationand further of the separation constituent of disaccharides the constituent/oligosaccharides disaccharides/oligosaccharides using sophisticated using techniques sophisticated including capillarytechniques electrophoresis including capillary [31,52,53 electrophoresis], HPLC [5,6,54 [31,52,53],,55], FACE HPLC (Fluorophore-Assisted [5,6,54,55], FACE ( CarbohydrateFluorophore- Assisted Carbohydrate Electrophoresis) [42,56] and mass spectrometry [57]. CS profile is achieved using Electrophoresis) [42,56] and mass spectrometry [57]. CS profile is achieved using well-established well-established multi-analytical laboratory techniques, particularly electrophoresis, HPSEC and multi-analytical laboratory techniques, particularly electrophoresis, HPSEC and eHPLC (Figure8)[ 5,6] eHPLC (Figure 8) [5,6] due to their high throughput, sensitivity and reproducibility. These due to their high throughput, sensitivity and reproducibility. These techniques, applied before and techniques, applied before and after enzymatic digestion of CS into smaller components, can after enzymatic digestion of CS into smaller components, can distinguish between CS derived from distinguish between CS derived from different animal or marine sources based on disaccharide diffcontent,erent animal patterns or marine of sulfation sources and based molecular on disaccharide size of the content, polymer patterns (Figure of 8) sulfation also using and analytical molecular sizestandards of the polymer of CS of (Figure different8) also origin using (see analytical below). However, standards a of big CS problem of di fferent remains origin for (see the below). sure However,identification a big problemof the origin remains of CS when for the mixed sure source identification material ofis theused origin to its production of CS when or mixed in the case source materialof possible is used cross-contamination. to its production or in the case of possible cross-contamination. The use of animal-derived sources to produce commercial CS also represents a concern for vegetarians and people with dietary restrictions related to religious and supply issues. In fact, Molecules 2019, 24, 1447 10 of 13 religious reasons and/or the practice of abstaining from the use of animal products have precluded the introduction of CS dietary supplements both in key emerging markets, such as Middle East and Asia, and in consolidated markets. The application of the above-illustrated multi-analytical techniques, electrophoresis, HPSEC and eHPLC requires highly qualified laboratories equipped with dedicated instrumentation and qualified technicians with specific analytical know-how producing very expensive activity and elevated costs for high quality CS management (Figure8). Moreover, an accurate and reproducible quantitative assay requires reference standards with high specificity, purity and well-known physicochemical properties and structures [6]. This is not a simple task in the case of CS due to its high heterogeneity of structure and properties mainly depending on the source and purification protocols, as previously discussed. However, several reference standards are commercially available, such as the CS standard from bovine cartilage manufactured by Bioiberica (www.bioiberica.com/) and possessing a purity greater than 98% and approved in 2004 as Chemical Reference Substance (CRS) by the European Pharmacopeia Commission [6]. Furthermore, other standards are available such as the European pharmacopoeia reference standard of CS sodium of marine origin (https://www.sigmaaldrich.com/) or the CS sodium from shark cartilage (https://www.sigmaaldrich.com/).

5. Conclusions In 2008, heparin manufactured in China was intentionally adulterated with an oversulfated CS polymer having a very high charge density quite similar to natural heparin, which provoked hundreds of deaths in Europe and USA after intravenous administration [58]. As for heparin, an important natural drug derived from animals, intentional adulteration of CS is difficult to detect since the tissue supply chain for CS in slaughterhouses generally lacks current good manufacturing processes (cGMP) oversight assuring high-quality CS production along with specific analytical quality controls. Moreover, as mentioned above, CS is derived from a variety of animal tissues potentially containing infectious agents leading to the transmission of viral, bacterial and prion diseases and the intentional or accidental mixing of CS from different animal species is highly suspected. In fact, some countries do not have a strict biosecurity system able to reduce the risk of the introduction and spread of disease agents. Even if specific and high-level analytical methods are generally able to distinguish from various sources, the detection of the presence of small amounts of different raw material remains very difficult. Additionally, CS structure can vary with environmental factors, animal subspecies, age and/or tissues providing additional difficulties for its quality and properties standardization. Moreover, the missing guidelines allow manufacturers of finished products to often change the sources of supply of the animal tissues or CS raw materials for competitive advantages. Consequently, the urgent solution to this scenario is the introduction and utilization in the current market of validated, specific, scientifically sound and largely published analytical controls. Manufacturers should ensure traceability of raw material right through to the finished product, with analytical evaluation made of each lot supplied to guarantee same quality, reproducibility and safety, as well as avoid misleading label information on finished products as reported in the literature. In conclusion, specific and accurate analytical procedures should be enforced for the control of high-quality products and applied by quality control laboratories to confirm the purity and label claims of CS in raw materials and finished products for food and nutraceutical applications. Finally, there are no definite regulations and common acceptance governing the origin of the ingredients in these supplements and the origin of the ingredients in products of natural and extractive origin is one of the most important factors ensuring quality, safety and efficacy.

Funding: This research received no external funding. Conflicts of Interest: The author declares no conflict of interest. Molecules 2019, 24, 1447 11 of 13

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