Identification of sulfation sites of metabolites and prediction of the compounds’ biological effects

Characterizing the biological effects of metabolic transformations (or biotransformation) is one of the key steps in developing safe and effective pharmaceuticals. Sulfate conjugation, one of the major phase II biotransformations, is the focus of this study. While this biotransformation typically facilitates excretion of metabolites by making the compounds more water soluble, sulfation may also lead to bioactivation, producing carcinogenic products. The end result, excretion or bioactivation, depends on the structural features of the sulfation sites, so obtaining the structure of the sulfated metabolites is critically important. We describe herein a very simple, high-throughput procedure for using mass spectrometry to identify the structure—and thus the biological fate—of sulfated metabolites. We have chemically synthesized and analyzed libraries of compounds representing all the biologically relevant types of sulfation products, and using the mass spectral data, the structural features present in these analytes can be reliably determined, with a 97% success rate. This work represents the first example of a high-throughput analysis that can identify the structure of sulfated metabolites and predict their biological effects.
Introduction
The study of drug metabolism is one of the critical steps in drug development [1–4]. At physiological conditions, drugs undergo a variety of biotransformations, which produce metabolites with different chemical structures. The resulting metabolites differ in both pharmacological and toxicological properties compared to their parent drugs [1]. Therefore, to identify and characterize the structures and properties of drug metabolites formed in vivo, an extensive investigation of the structures generated by biotransformation is required.
Sulfate conjugation (sulfation) is one of the most important phase II reactions that occurs during the biotransformation of a variety of structurally diverse endogenous compounds, xenobiotics, and drugs [5–9]. During this process, a sulfonate moiety (SO3) is transferred from the donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the substrates through the catalysis of sulfotransferase enzymes, resulting in sulfated products [10]. Sulfation is generally considered as a detoxification pathway, because the sulfated products are more water-soluble; this facilitates their elimination from the body [11–13]. However, sulfation can also lead to the bioactivation of certain type of compounds which include benzylic, allylic alcohols and aromatic hydroxylamines. Sulfated products of these compounds can undergo loss of , resulting in reactive electrophilic carbocation or nitrenium ion intermediates, which covalently bind to cellular macromolecules, leading to mutagenicity and carcinogenicity [6, 14, 15]. The production of the electrophilic intermediates relies on the structural feature of sulfation sites, as shown in Fig. 1. Under physiological conditions, the sulfate anions in substrates a–c in Fig. 1 act as good leaving groups to form resonance-stabilized intermediates [6, 14]. For the other types of sulfated products in Fig. 1, this process is not favorable due to instability of cation products.
Fig. 1Mechanism of forming bioactive carbocation or nitrenium ion intermediates from sulfated products. Substrates a, b and c undergo the bioactivation process while d–g do not. a sulfated benzylic alcohol; b sulfated allylic alcohol; c sulfated aromatic hydroxylamine; d) sulfated aliphatic alcohol; e sulfated phenol; f sulfated aliphatic amine; g sulfated aromatic amine
The biological sulfation of tamoxifen is one example that demonstrates how the sulfation site dictates the biological fate of the molecule. Tamoxifen is a pharmaceutical that is widely used in the treatment and prevention of breast cancer. It is hydroxylated during phase I metabolism to generate two products: 4-hydroxytamoxifen (4-OH-TAM) and alpha-hydroxytamoxifen (α-OH-TAM). The sulfation of 4-OH-TAM and that of α-OH-TAM have completely different biological effects. The sulfation of 4-OH-TAM, which occurs at a phenol site (as in Fig. 1), leads to detoxification, whereas the sulfation of α-OH-TAM, which involves sulfation of a benzylic alcohol (as in Fig. 1a), leads to bioactivation [16, 17]. This example (along with many others) demonstrates the fact that the biological effects of sulfation can be determined by characterizing the sulfation site of the substrate.
It is widely known that aromatic or aliphatic hydroxyl and amine groups are the major sites in molecules that can be sulfated during metabolism [18]. However, identifying the sulfation sites can be difficult because many potential sulfation sites can be produced or modified during phase-I biotransformations [5]. For example, hydroxyl groups can be added to a benzene ring or carbon chain through aromatic or aliphatic hydroxylation, generating new potential sulfation sites as in the tamoxifen example. An amine group can be changed to a hydroxylamine group through N-oxidation [1], which modifies the structural feature of the potential sulfation site. Since these new sites are formed in vivo, characterizing the biological effect of their sulfation can be problematic. It is thus essential to develop a method that can characterize the sulfation sites of unknown sulfated metabolites. With this information, the biological effect of sulfation can be estimated, and the properties of sulfated metabolites can be characterized.
Analytical techniques such as NMR spectroscopy and mass spectrometry have been utilized to identify sulfated metabolites [19–31]. NMR is a powerful technique that can not only detect sulfated metabolites [20, 21, 24], but also help to determine the position of sulfation in the substrate molecules [19, 22, 23]. However, due to the relatively high sample requirements, the complexity of data analysis, and the need to characterize purified compounds, the application of NMR in identification and characterization of drug metabolites is limited in high-throughput metabolite profiling. Compared to NMR, mass spectrometry is more widely used in drug metabolite identification, due to its high selectivity and sensitivity, low detection limit, and ability to analyze mixtures [32]. It is known that mass spectrometry can identify sulfation by detecting the 80 Da mass increase in MS mode, or the characteristic ions in MS/MS mode [21, 25–31]. However, limited information about structural features of sulfation sites currently can be obtained from MS/MS analysis, with the well-known exception that the product ion m/z 97 () can be used to differentiate alicyclic sulfates from aromatic sulfates [25, 26].
In order to facilitate the identification of sulfation sites in pharmaceuticals, we developed a mass spectrometry-based protocol that differentiates between the biologically relevant sulfation sites. After completing the studies on several classes of sulfated products, a set of rules was developed to predict the sulfation sites. With these prediction rules, structural feature of sulfation sites can be determined by detecting MS/MS fragmentation pathways of their corresponding sulfated products. With the structural information of sulfation sites, the two key different biological effects of sulfation, “detoxification” and “bioactivation”, can be differentiated, based on literature precedence that links the type of sulfation to its biological fate. Based on that information, relevant properties of sulfated products can be estimated.
Experimental
Reagents Sulfates and sulfamates which include 4-nitrocatechol sulfate dipotassium salt, L-ascorbic acid 2-sulfate dipotassium salt, indoxyl sulfate potassium salt, β-estradiol 3-sulfate sodium salt, potassium 4-nitrophenyl sulfate, β-Estradiol 3-sulfate sodium salt, 5-Br-4-Cl-3-indolyl sulfate potassium salt, 4-methylumbelliferyl sulfate potassium salt, 2-aminoethyl hydrogen sulfate, poly (vinyl sulfate) potassium salt, D-glucose 6-sulfate potassium salt, chondroitin disaccharide Δdi-6S sodium salt, N-acetylglucosamine 6-sulfate sodium salt, N-cyclohexylsulfamic acid, 3-hydroxypropyl-sulfamic acid monopotassium salt, D-glucosamine 2-sulfate sodium salt, and butyl-sulfamic acid were purchased from Sigma-Aldrich (St. Louis, MO). One sulfamate, 4-methylphenyl- sulfamic acid, was purchased from Scientific Exchange, Inc. (Center Ossipee, NH). (R)(+)-α-phenethylsulfamic acid was purchased from Norse Laboratories (Newbury Park, CA).The benzylic alcohols, allylic alcohols, hydroxylamines which include (R)-(+)-α-methyl-2-naphthalenemethanol, benzyl alcohol, (R)-1-phenyl-2-propen-1-ol, 4-chloro-2-methylbenzyl alcohol, 3-ethoxybenzyl alcohol, 2-ethoxybenzyl alcohol, 4-ethoxybenzyl alcohol, crotyl alcohol, furfuryl alcohol, N-methylhydroxylamine hydrochloride, N-isopropylhydroxylamine hydrochloride, N-cyclohexylhydroxylamine hydrochloride, N, N-diethylhydroxylamine and N-benzoyl-N-phenylhydroxylamine were purchased from Sigma-Aldrich (St. Louis, MO). These benzylic alcohols, allylic alcohols, and hydroxylamines were utilized to synthesize their corresponding sulfated products. The other reagents used in the sulfation of alcohols and hydroxylamines were also purchased from Sigma-Aldrich (St. Louis, MO).
Sulfation of benzylic, allylic alcohols and tertiary hydroxylamines Sulfated products were prepared based on slight modifications to a previous protocol [33] by dissolving 1.0 equivalent (5.0 mmol) of the substrate in 5.0 ml dimethylformamide (DMF) and adding sulfur trioxide-dimethylformamide complex (DMF-SO3 5.5 mmol) and pyridine (5.5 mmol). The reaction mixture was stirred at 40 °C for 1 h. H2O (35 ml) was added to the product solution, and the product was extracted with 3×15 mL ethyl acetate (EtOAc). Evaporation of the solvent afforded the corresponding sulfated product.
Sulfation of secondary hydroxylamines To a dry THF (10 ml) solution of NaH (10.0 mmol) was added secondary hydroxylamines (5 mmol) at 0 °C. Then the solution was stirred at room temperature for 2 h, followed by reaction with DMF-SO3 (5.0 mmol) overnight. The corresponding sulfated products were obtained by evaporation of THF.
Sample pretreatment Sulfated compounds that were purchased were dissolved to a final concentration of 1.0×10−4 M with 50% MeOH/ 50% H2O, and directly injected into the mass spectrometer. Sulfated benzylic and allylic alcohols and tertiary hydroxylamines were prepared by diluting 10 μl of the liquid product by 100 fold with 50% MeOH/50% H2O, and then injected to the mass spectrometer. Sulfated secondary hydroxylamines were prepared by dissolving 10 mg solid product in 1.0 ml 50% MeOH/ 50% H2O, followed by injection into the mass spectrometer.
Mass spectrometry The mass spectra were recorded using a Quattro Ultima (Waters Corp., Milford, MA) triple quadrupole mass spectrometer equipped with an electrospray ionization source. Data was acquired in the negative ion mode using a capillary voltage of 2.80 kV and a cone voltage was 45 V. The source temperature and desolvation gas temperature were 80 and 150 °C, respectively. Argon is used as the collision gas, and the pressure in collision cell is 1.7E(−3) mbar. The collision energy applied in MS/MS for all the samples was 35 eV.
Results and discussion
The structures of the sulfated products are in Fig. 2. These compounds are representative of different types of sulfated products that are produced by sulfation at different sites. Mass spectrometric studies were conducted in the negative ion mode since sulfated products are deprotonated at physiological pH [11, 15]. The common product ions from the MS/MS experiments are summarized in Table 1. As the table indicates, common product ions include m/z [M-H-80], m/z 80, m/z 96 and m/z 97. The presence of m/z [M-H-80], m/z 96 or m/z 97 ions are quite dependent on the site of sulfation. The presence of the negative ion with m/z 80, however, is common in the fragmentation of almost every type of sulfated products, so it is less helpful in identification of sulfation sites. Thus this ion, m/z 80, is used to identify sulfation sites when no other characteristic ions can be detected. Based on the MS/MS data, each type of sulfated product possesses a characteristic fragmentation pathway, which is described below. An example of MS/MS data for each type of sulfated product is provided in Fig. 3 with the characteristic ion labeled by an asterisk.
Fig. 2Structures of selected sulfated compounds a sulfated aromatic alcohols or enols; b sulfated aliphatic alcohols with β hydrogens on sp3carbons; c sulfated benzylic or allylic alcohols; d sulfated hydroxylamines; e sulfated aminesFig. 3Characteristic (-) ESI-MS/MS data for compounds in this study: a sulfated aromatic alcohol, from group a; b sulfated aliphatic alcohol with β hydrogen on sp3carbon, from group b; c sulfated benzylic alcohol, from group c; d sulfated aliphatic hydroxylamine, from group d; e sulfated aromatic hydroxylamine, from group d; f sulfated aliphatic amine, from group e; g sulfated aromatic amine, from group eTable 1Characteristic fragmentation of selected sulfated productsGroupNo.Chemical nameAbundance of characteristic ions in MS/MS (%)m/z [M-80]-m/z 80m/z 96m/z 97a14-nitrocatechol sulfate100a–––2L-ascorbic acid 2-sulfate31a–––3Indoxyl sulfate24a52––44-nitrophenyl sulfate100a–––5β-estradiol 3-sulfate100a–––65-Br-4-Cl-3-indolyl sulfate47a59––74-methylumbelliferyl sulfate100a–––b82-aminoethyl hydrogen sulfate –1001867a9Poly (vinyl sulfate)–––97a10D-glucose 6-sulfate–––100a11Chondroitin disaccharide Δdi-6S –––31a12N-acetylglucosamine 6-sulfate–––93ac132-naphthalenemethanol, α-methyl- sulfate–6.77.3a10014Benzyl sulfate–16100a–15(R)-1-phenyl-2-propen-1-sulfate–26100a12164-Cl-2-methylbenzyl sulfate––100a95173-ethoxybenzyl sulfate–10013a6.4182-ethoxybenzyl sulfate–10017a8.5194-ethoxybenzyl sulfate–10020a8.720crotyl sulfate–5591a5021furfuryl sulfate––53a39d22N-methyl-hydroxylamine-O-sulfonic acid–10073a9.223N-isopropyl- hydroxylamine-O-sulfonic acid–68100a5.124N-cyclohexyl-hydroxylamine-O-Sulfonic acid–27100a1125N,N-diethyl-hydroxylamine-O-sulfonic acid–92100a1926N-benzoyl-N-phenyl-hydroxylamine-O-sulfonic acid––100a–e27N-cyclohexylsulfamic acid–100a––284-methylphenyl-Sulfamic acid–100a––293-hydroxypropyl-sulfamic acid–100a––30D-glucosamine 2-sulfate–100a––31butyl-sulfamic acid–100a––32(R)(+)-α-phenethylsulfamic acid–100a––*A threshold of 5.0% is used for the relative abundance of characteristic ions. The long dash (–) means that the characteristic ion can not be detected or the relative abundance is below 5.0%aRepresents the specific characteristic ion of each group that can help to identify the sulfation site
Group a: sulfated aromatic alcohols or enols
Compounds 1–7 are sulfated aromatic alcohols or enols. For these compounds, the sulfate group is attached to an sp2 carbon (benzene ring or double bond). These sulfated products share the same fragmentation pathway by undergoing the neutral loss of 80 Da, and forming a characteristic ion at m/z [M-H-80], as seen in Fig. 3a. After undergoing the neutral loss of SO3, phenoxide or enolic anions are formed, and the negative charge is resonance stabilized, as shown in Scheme 1 [33].
Scheme 1Sulfated aromatic alcohols or enols (group a) dissociate to produce a characteristic ion with neutral loss of 80 Da. Group b: sulfated aliphatic alcohols with β hydrogens on sp3carbons
The second group of sulfated products originates from the sulfation of aliphatic alcohols: compounds 8–12. For these species, the sulfate group is attached to an sp3 carbon. All sulfated products of this type can produce the characteristic ion m/z 97 in MS/MS, and one example is shown in Fig. 3b. The ion m/z 97 is generated when the proton from the β carbon is transferred to the sulfate moiety, and the C-O bond is broken to form a bisulfate anion [33]. See Scheme 2.
Scheme 2Sulfated aliphatic alcohols (group b) produce the characteristic ion m/z 97
This rearrangement occurs via an energetically accessible six-membered ring transition state, and a stable neutral product is formed. As a result, this fragmentation is predominant in the MS/MS data of this group of sulfated products. The mechanism shows that the availability of a β hydrogen on an sp3 carbon is required for this fragmentation. Unlike compounds in group a, neutral loss of 80 Da is not observed from this group of compounds. This is likely due to the fact that the product ion that would be generated from loss of 80 Da (SO3) is not resonance stabilized, so the loss is not favorable. Compound 8 is a special case because it gives a characteristic ion with m/z 96, in addition to m/z 97. The relevant mechanism for this loss is addressed in the discussion of compounds in group d.
Group c: sulfated benzylic or allylic alcohols
In the third group of sulfated compounds (13–21), each contains a sulfate group attached to benzylic or allylic carbon. All the compounds in this group produce a characteristic ion with m/z 96. An example of an MS/MS data for this type is shown in Fig. 3c. The characteristic ion is produced by homolytic cleavage, producing benzylic or allylic radicals. The radical is resonance stabilized, which is demonstrated in Scheme 3 [33].
Scheme 3Sulfated benzylic and allylic alcohols (group c) produce the characteristic ion m/z 96
In addition to m/z 96, the negative ion m/z 97 can also be obtained for some of the compounds in this category. For example, compounds 13 and 16 both have an abundant peak at m/z 97, due to the availability of a hydrogen on the sp3 carbon in close proximity to the sulfate group. Even though compound 13 is a benzylic sulfate, a hydrogen attached to the β sp3 carbon is present. Therefore, just like compounds in group b, it undergoes a rearrangement to form the ion, m/z 97. Compound 16 possesses a methyl group in the ortho position, and the availability of hydrogen attached to the sp3 carbon in methyl group enables a similar rearrangement (eight-membered ring) to form the product ion, m/z 97, as illustrated in Scheme 4. Even though β hydrogens are available in other benzylic and allylic sulfates in this group, the fragmentation to produce m/z 97 is not as favorable, since the hydrogens are on the sp2 hybridized carbons. The neutral loss of 80 Da is not favorable either, because the product ion that would be generated from such a loss is not resonance stabilized.
Scheme 4Fragmentation mechanism producing m/z 97 from compound 16
Group d: sulfated hydroxylamines
The fourth group of sulfated products originates from sulfation of hydroxylamines, and as a result, the sulfate group is directly attached to the amine: See compounds 22–26. All the sulfated products of this group can produce the characteristic ion m/z 96, and examples of MS/MS data are shown in Fig. 3. A characteristic ion with m/z 96 can be obtained for sulfated products originating from both aliphatic (Fig. 3d) and aromatic (Fig. 3e) hydroxylamines. For these sulfated compounds, homolytic cleavage is favorable. This is due to the small electronegativity difference (about 0.5) between N and O, compared to electronegativity difference of around 1.0 for C –N and O-S [34]. The small electronegativity difference causes the electrons to be split equally (homolytic cleavage) between the oxygen and nitrogen when the bond breaks, instead of both electrons moving onto the oxygen or nitrogen, which would be the case during heterolytic cleavage. In Scheme 5, the mechanism of this homolytic cleavage is depicted. An amine radical is formed as the product.
Scheme 5Sulfated hydroxylamines (group d) produce the characteristic ion m/z 96
This mechanism can also be used to explain the presence of the ion with m/z 96 in the MS/MS spectrum of compound 8. Scheme 6 shows how this ion is produced from compound 8. In this case, homolytic cleavage can be used to distribute a single electron onto the nitrogen. Compounds that contain a nitrogen two carbons away from the sulfation site, like this one, could also produce m/z 96 as a product ion.
Scheme 6Fragmentation mechanism producing m/z 96 from compound 8
Group e: sulfated amines
The last group of sulfated products, sulfamates, originates from the sulfation of amines. Compounds 27–32 belong to this type. The only characteristic ion for this type of sulfated products is the negative ion, m/z 80. This characteristic ion can be obtained from fragmentation of sulfated products originating from both aliphatic amines (Fig. 3f) and aromatic amines (Fig. 3g). This group of sulfated products has another type of bond, a N-S bond, that has a small electronegativity difference between the two atoms (0.5) [34], making the homolytic cleavage between the nitrogen and sulfur favorable. The mechanism that shows production of m/z 80 is illustrated in Scheme 7. No other fragmentation pathway is as favorable as this homolytic cleavage pathway, making m/z 80 the predominant ion in all of the MS/MS data for these compounds.
Scheme 7Sulfated amines (group e) produce the characteristic ion m/z 80
Prediction rules
For each type of sulfated product, there is a predominant fragmentation pathway that results in a characteristic product ion during MS/MS fragmentation. The specific characteristic ion for each group is obtained from every sulfated product in that group, as shown in Table 1: the ion with the neutral loss of 80 Da can be seen in MS/MS spectrum of every sulfated product in group a; an ion with m/z 97 can be detected in all the MS/MS data of group b compounds; an ion with m/z 96 can be obtained from MS/MS data of all the sulfated products of both group c and group d. A negative ion with m/z 80 can always be found in MS/MS data of sulfated products in group e.
We have shown that characteristic fragmentation pathways of sulfated products are dependent upon the structural features of the sulfation sites. Conversely, relevant structural information of sulfation sites can be obtained by determining characteristic fragmentation pathways or characteristic ions. To facilitate this information transformation, a set of prediction rules is described as follows:
If the characteristic ion with a neutral loss of 80 Da can be detected, this indicates that the sulfate group is attached to an sp2 carbon, the sulfated product is therefore produced by the sulfation of a phenol or an enol.If the characteristic ion with m/z 96 can be detected, the sulfated product is produced by sulfation of a benzylic or allylic alcohol, or a hydroxylamine.If the characteristic ion with m/z 97 can be obtained, and it is more abundant than the ion, m/z 96 (if it is detected), the sulfate group is attached to an sp3 carbon. This is the sulfated product produced by sulfation of aliphatic alcohols with an available β hydrogen attached to an sp3 carbon;If none of the ions, m/z [M-H-80]−, m/z 96, or m/z 97 can be detected, and only the ion m/z 80 is observed, then the sulfated product is produced by sulfation of an amine.
There are occasions when the conditions in rule 2 and 3 might both be observed. For example, both of these apply to compound 13, which suggests that the sulfation site is not only an alcohol with available β hydrogen (on an sp3 carbon), but also a benzylic or allylic alcohol; and this estimation is consistent with the actual structure of compound 13. Among the selected 32 sulfated products, 31 of them follow the proposed prediction rules. The exception, compound 8, possesses an amine group in the β position. The amine at this position enables the compound undergo the fragmentation pathway to produce ion with m/z 96 (see Scheme 6). In this case, rule 2 incorrectly predicts the sulfation site for compound 8. As a result, the obtained data in Table 1 demonstrates a 97% (31/32=0.97) success rate of these prediction rules.
Potential application of the prediction rules in characterizing unknown sulfated metabolites
As one of the major phase II biotransformations, sulfate conjugation is involved in the metabolism of an enormous range of substrates [5]. In general, sulfation is a detoxification or deactivation pathway, but it can also bioactivate the substrate molecules when sulfation sites are benzylic, allylic alcohols (group c) or aromatic hydroxylamines (group d) [6, 8, 14]. According to the prediction rules developed herein, sulfation at any of these sites will generate products that produce the ion m/z 96 in MS/MS, so the structural feature of the sulfation sites can be determined by rule 2. Sulfation of other sites, like phenols, aliphatic alcohols or amines, will cause other characteristic ions to appear in MS/MS data. Therefore, the sulfation sites can be determined by rule 1, 3 or 4. In these cases, sulfation facilitates detoxification.
With the proposed prediction rules, biological effects of sulfation can be determined, and the properties of sulfated metabolites can be estimated. Figure 4 demonstrates how the proposed method works to characterize unknown sulfated metabolites. Since high-quality MS/MS data is a necessary prerequisite of using this method, if the metabolite is present in a complex biological matrix, some mass spectral optimization may be necessary. After obtaining reliable (-)ESI-MS/MS data on the unknown sulfated metabolite, the prediction rules are used to determine sulfation site by identifying the characteristic ions present in the MS/MS data. If the MS/MS data matches rule 1, 3 or 4 (instead of rule 2), the sulfation sites can be phenols (or enols), aliphatic alcohols (with β hydrogen on sp3 carbon) or amines. In these cases, the sulfation undergoes a detoxification pathway and the sulfated metabolite is more easily eliminated, compared to the parent drug. If the MS/MS data is consistent with rule 2, the sulfation site should be benzylic, allylic alcohols or hydroxylamines. This would indicate that the sulfation might follow a bioactivation pathway, and lead to carcinogenic sulfated products. One potential caveat to this second condition is that a few nontoxic sulfated metabolites that have structures similar to compound 8, might also produce the characteristic ion with m/z 96, following the mechanism in Scheme 6. Another possible drawback of this characterization is that the diagnostic ion m/z 96 does not discriminate between sulfated products of aliphatic and aromatic hydroxylamines; and thus far only aromatic hydroxylamines have been proven to be toxic. However, distinguishing between aromatic and aliphatic hydroxylamines is not critically important because sulfated aliphatic hydroxylamines are rather uncommon metabolites. While it is possible that a few compounds could be misassigned as “toxic”, none of the sulfated metabolites that are known to be bioactivated would be misdiagnosed as “nontoxic”, as demonstrated herein.
Fig. 4The method for characterizing unknown sulfated metabolites
Conclusion
A method was developed to determine the structural features of sulfation sites, by detecting the characteristic fragmentation pathway of the corresponding sulfated products in (-) ESI-MS/MS. By summarizing MS/MS data from five different types of sulfated products originating from different sulfation sites, their characteristic fragmentation pathways and characteristic ions were determined. Based on this information, a set of prediction rules was developed to transfer information about the fragmentation pathway of sulfated products to the structural features of the sulfation site. As a result, the proposed prediction rules can be applied in drug metabolite profiling to characterize sulfation sites, to further estimate the biological effect of sulfation, and to evaluate relevant properties of sulfated metabolites.

sulfation
metabolites
pharmaceuticals
mass spectrometry
icp-ms
bioanalytical methods
biological samples


Neutron activation analysis and X-ray Rayleigh and Raman scattering of hair and nail clippings as noninvasive bioindicators for Cu liver status in Labrador Retrievers

The heritability of chronic hepatitis in the Labrador Retriever is studied with the aim of identifying the related gene mutation. Identification of cases and controls is largely based on instrumental neutron activation analysis (INAA) Cu determination in liver biopsies. The burden for these companion animals may be reduced if nail clippings and hair (fur) could serve as a noninvasive indicator for the hepatic Cu concentrations. No correlation was found between hepatic Cu concentrations and Cu concentrations in hair and nail samples. However, hair and nail samples were also analyzed by X-ray tube excitation, taking advantage of the X-ray Compton, Rayleigh, and Raman scattering which reflects the organic components such as the type of melanin. Principal component analysis provided first indications that some differentiation between healthy and sick dogs could indeed be obtained from hair and nail analysis.
Introduction
Chronic hepatitis in dogs is a histological diagnosis, characterized by the presence of fibrosis, inflammation, and necrosis of the liver cells. Symptoms of chronic hepatitis are quite variable but the most common signs are lethargy, loss of appetite, and diarrhea. Pets may also drink and urinate more. As the disease progresses, many dogs develop yellowish gums, eyes, and skin (icterus/jaundice), and a swollen abdomen which is filled with fluid (ascites). In some cases toxins affect the nervous system and the dogs become blind and obtuse. This can progress to seizures, coma, and death. The disease is characterized, amongst others, by copper accumulation in the liver. Previous studies have shown elevated hepatic Cu concentrations in the order of 1,000–2,000 mg kg−1 compared with normal Cu levels of < 200–500 mg kg−1. Such hepatic copper accumulation can result from increased uptake of copper, a primary metabolic defect in hepatic copper metabolism, or from altered biliary excretion of copper. Several studies have already been conducted at Utrecht University on inherited copper toxicosis and hepatic copper storage in companion dogs such as Bedlington Terriers [1] and Dobermanns [2]. Currently, one of the programs is investigating the genetic basis of the disease by examination of family members from Labrador Retrievers with copper-associated chronic hepatitis.
The selected dogs undergo blood sampling, and retrieval of a liver biopsy (by use of the Menghini technique). The liver biopsy is examined histologically and stained for copper granules. As this project deals with companion animals from the Labrador population in The Netherlands and not with test animals, cooperation of the dog owners is crucial. Following the Delft group’s involvement and over 20 years’ experience investigating nail clippings as a bioindicator for the trace element status in man [3, 4], a feasibility study was undertaken to assess the usefulness of nail clippings and hair (fur) as a noninvasive indicator for the Cu liver levels with the aim of replacing the transcuteaneous full-needle biopsy.
Two approaches were tested. The most straightforward was to verify if Cu levels in nail clippings and hair would reflect the Cu liver levels. The other approach was initiated by the observation that a copper deficiency may result in hair/coat color changes. This is ascribed to the activity of cuproenzymes, which catalyze the biosynthesis of melanin from L-tyrosine. A copper deficiency causes, among others, pigmented hair on the head and face to lose its normal color and get a “washed out” appearance and become gray [5].
In 2005, Bueno et al. demonstrated that X-ray Rayleigh peaks and especially the peakless part in the X-ray Raman scattering continuum in X-ray spectra obtained by tube excitation carry information on the organic constituents of biological materials [6, 7]. A study of Poodles in which all spectral information was processed by dedicated principal component analysis (PCA) resulted in a clear differentiation of all dogs examined on the basis of their hair color and, moreover, the separation of healthy and sick dogs on the basis of the hair analysis. Hair pigmentation is due to two major types of melanin, viz., eumelanin and phaeomelanin (Fig. 1). The type of melanin manifesting in the hair can be attributed to the hair color genotype [8, 9], whereas the intensity and distribution of the granules may also be affected by diseases in which sulfur metabolism is involved.
Fig. 1Chemical structures of eumelanin and phaeomelanin
Both approaches were tested here, i.e., (i) Cu determination via instrumental neutron activation analysis (INAA) in liver biopsies, nail clippings, and hair samples; and (ii) X-ray spectrum acquisition after X-ray tube excitation, followed by full spectrum data (i.e. all channel contents) processing by PCA, including the often neglected peakless part of the X-ray scattering region resulting from Compton, Rayleigh and Raman effects.
Experimental
Liver biopsy samples, nail clippings, and hair were collected from approximately 100 dogs with chronic hepatitis as well as from healthy animals. Nail clippings were taken from phalanx I (the thumb nail), since this nail is not in contact with the ground, thus minimizing contamination problems. Nail clippings could not be collected from all animals as in some cases breeders had removed the nail permanently shortly after birth, since sometimes such a thumb nail hinders the dog in working trials. Hair was sampled from the median abdominal wall, just cranial to the belly button.
The liver biopsy samples were freeze-dried at the veterinary clinic. The nail clippings and hair samples were analyzed without cleaning to prevent changes in the nail and hair morphology that might affect the scattering information from the X-ray fluorescence (XRF) analysis. It should be noted that a Cu contamination of the nail clipping and hair is highly unlikely; the nail of the thumb nail is never in contact with, e.g., soil, and external contamination of the hair was also unlikely.
INAA
The needle biopsies were transferred into polyethylene capsules and lyophilized, resulting in sample masses varying typically between 1.5 and 50 mg dry weight. Nail clippings had masses of 5–50 mg, and the typical mass of the hair samples was approximately 100–200 mg.
The Cu levels were established by INAA via the determination of 66Cu. This is the radionuclide of choice although 64Cu is also produced; however, the associated 511-keV peak is insufficient for reliable Cu determination, whereas the 1,345-keV peak provides insufficient sensitivity. Quantification of all element mass fractions was based on the single comparator method [10]. Samples were irradiated one-by-one for 3 min in the Hoger Onderwijs Reactor in a thermal neutron fluence rate of 1.7 × 1017 m−2 s−1. A Zn flux monitor was included in each irradiation; the neutron fluence rate was determined by measurement of the induced 69mZn activity. The activated biopsy, nail, and hair samples were measured 30 s after irradiation for 3 min at a 1-cm distance from the end-cap of a side-looking 12% Ge detector. The samples were rotated during counting. The Zn monitors were measured in the same geometry for 60 s after a 4-min decay time. The samples were processed in batches of 14 samples, one internal quality control sample, and a blank. A sample of NBS 1577b Bovine Liver (certified Cu content 160 ± 8 mg kg−1) was used for internal quality control. Details of the irradiation facility, gamma-ray spectrometer, and associated quality assurance have been published elsewhere [11]. The spectrometer is equipped with ORTEC DSPEC-PLUS electronics, which allows for adequate dead-time correction [12]. It is obvious that, particularly in the nail and hair samples, several other elements such as Ca, Mg, and S were also quantified on the basis of their short halflife radionuclides, measured simultaneously with 66Cu.
X-ray tube excitation
The measuring procedure consisted of weighing approximately 100 mg of each hair sample and 10 mg of each nail sample directly into appropriate cells and submitting them to blank rhodium X-ray tube radiation, in triplicate. A common laboratory energy dispersive XRF instrument (EDX 700, Shimadzu) was used under the same irradiation conditions: 50-kV applied voltage in the tube, 25% dead time, 10-mm beam collimation, and 100-s irradiation time. It should be noted that the methodology of using the region in the X-ray spectrum resulting from scattering implies that only the spectrum’s shape is needed for the data processing by PCA. There is no need for quantification towards Cu mass fractions, and hence no reference materials were used for quality control as was the case in the hair, nail, and liver biopsy analysis by INAA.
The chemometric method [13] used for data analysis was PCA, processed via the software The Unscrambler, version 9.2, from Camo.
Results and discussion
Cu could not be determined in all available nail samples because of the small sample masses and the detection limit of the analytical protocol selected. Detection limits on the basis of 66Cu were typically in the order of 25 ng, corresponding to a minimum detectable Cu mass fraction of 5 mg kg−1 in a 5-mg nail clipping sample. Cu levels in hair and nail are given in Fig. 2, together with the associated Cu liver levels. Uncertainties are not depicted for clarity reasons; typical uncertainties (combined standard uncertainty) were approximately 5–20% for the liver analyses, 15–30% for the nail, and 5–10% for the hair analyses. It is clear from Fig. 2 that the Cu levels in hair and nail do not reflect the Cu liver levels. This indicates that the gene(s) responsible for the Cu accumulation in the liver is(are) only active in the liver cells and not in the cells at the nail base or in the hair pocket.
Fig. 2Cu levels in nail clippings and hair of Labrador Retrievers as a function of the Cu levels in liver biopsies taken from the same dogs: ○ hair, ● nail; uncertainties are omitted for clarity (see text)
Principal component analysis (PCA) results of the processing of the channel contents of the X-ray spectra of all hair samples are given in Fig. 3, and the related factor loadings of PC1 and PC2 are shown in Fig. 4 (similar results were found for the processing of the channel contents of the X-ray spectra of the nail samples, although these are not shown here). The PC1 loading graph indicates the dependent variables that explain most of the data variance, and the PC2 loading graph indicates variables that explain the second data variance. PC1 and PC2 are orthogonals and therefore independent of each other.
Fig. 3PCA analysis of all hair samples, classified here by the hair color: □ yellow, * chocolate, ■ blackFig. 4PC1 loadings for hair samples (top). PC2 loadings for hair samples (bottom)
The variables included in the PCA analysis were from the channel contents between 0 and 40 keV, i.e., covering the entire spectum recorded.The symbols in Fig. 3 reflect the hair color. The differentiation with respect to coat color results most likely from differences in type of melanin in Labradors with a black, liver (chocolate), or yellow-colored coat. Table 1 shows the genotypes of the hair color inheritance of Labrador Retrievers [8], in which each character represents a specific gene (denoted in pairs, of course). An uppercase character represents a dominant gene, a lowercase character represents a recessive gene; no character in a gene pair implies that it is not relevant for the genotype if the locus is occupied by a dominant or recessive gene. As produces black without any tan on the dog and there are other genes that can modify the black to liver (chocolate Labrador). If As is present, in most cases the dog will be able to produce only eumelanin pigment. Some shades of liver (chocolate), though a eumelanin pigment, overlap some shades of tan, a phaeomelanin pigment. The e, recessive red, overrides whatever gene is present at the A locus to produce a dog which shows only phaeomelanin pigment in the coat. The related melanin types are also included in Table 1 and the color differentiation by the principal component analysis is most probably based on the differences in melanin corresponding to the different genotypes. The wide spread of the groups might reflect homozygotes and heterozygotes for the genes involved.
Table 1Labrador genotypes [8] for hair color inheritance and corresponding types of melaninColorGenotypeMelanin typeBlackAs–  B–  E–EumelaninLiver (chocolate)As–  bb  E–EumelaninYellow, liver nose– –  B–  eePhaeomelaninYellow, black nose– –  bb  eePhaeomelanin
The PCA also revealed a differentiation to gender, as is further exemplified in Fig. 5 for nails. This is in agreement with earlier work for Poodles in which a similar gender separation was observed [6].
Fig. 5PCA analyses of all nail samples, classified by gender
These results (Figs. 3, 4, 5) indicate that PC1 allows for a separation of dogs by gender and PC2 for separation by color, particularly when using hair samples. The loading graphs of the PCs (Fig. 4) show that sulfur and Rh scatter (Compton, Rayleigh, and Raman) explain the variance in PC1. This is most likely attributed to variations in (sulfur bondings in) keratin structures. This effect contributes to almost 100% of the data variance. The rest of it, PC2, is not very large (approximately 1–10%) and allows for separations due to differences in sulfur, and to some extent with melanin.
The physiological cause of the variation of the keratin structures in hair and nail by gender is not yet fully clear, but there are several indications that the structure of hair is different for males and females. It is well known that there is a relationship between testosterone and hair formation (andropogenic hair). Andropogenic hair has a different growth rate and a higher weight than other hair. It has also been observed that the type of sulfur is different for females and males: rhombic (alpha) sulfur predominates in female hairs, monoclinical (beta) sulfur in male hairs [14]. Less information is available about nails. Genetic variation in the proteins of the human nail has been observed [15], as well as a sex variation in lipid composition of human fingernail plates [16].
Another possible explanation for the separation of dogs in terms of genders in the hair analysis might be found in the results of Scott et al. [17] who concluded that the human melanocortin 1 receptor (MC1R) is regulated by, amongst others, specific endocrine sex hormones and by UV radiation. In confirmation of the latter study, Broekmans et al. [18] found that male subjects were more sensitive to UV irradiation than female subjects. It was already known that eumelanin is photoprotective, whereas phaeomelanin may contribute to UV-induced skin damage due to its potential to generate free radicals in response to UV radiation [19]. Skin melanin content, which was positively associated with hair color in men, was the main phenotypical determinant of sensitivity to UV irradiation.
The PCA results could also be plotted as a function of the INAA Cu liver biopsy values. Results for hair and nail are given in Fig. 6 for a differentiation towards dogs with Cu liver levels of < 600 mg kg−1 or > 600 mg kg−1. A Cu biopsy level of > 600 mg kg−1 is considered to be positively indicative for liver Cu accumulation. In addition, plots were also made in which the hair and nail samples are identified by correlating with Cu biopsy values > and < 200 mg kg−1 as well as at > and < 400 mg kg−1. At these lower Cu levels no clear differentiation is obtained, as could be expected, which confirms that the PCA is not producing an artifact, accidentally coinciding at the 600 mg kg−1 Cu liver biopsy level.
Fig. 6PCA results, nail X-ray spectral data vs. INAA Cu liver biopsy values (top): ■ dogs with Cu liver levels 0–600 mg kg−1, □ dogs with Cu liver levels > 600 mg kg−1 , N = 60. PCA results, hair X-ray spectral data vs. INAA Cu liver biopsy values (bottom): ■ dogs with Cu liver levels 0–600 mg kg−1, □ dogs with Cu liver levels > 600 mg kg−1, N = 43
In all PCA, it could be justified to remove some outliers on the basis of high leverage values and high energy variance residuals in PCs. High values of leverage and residuals are caused, for instance, by imperfect quality and positioning of the sample during the X-ray irradiation. In addition, outliers may sometimes result from the presence of dogs with strong parental relationships [6].
The strength of using the information of the scattered radiation is emphasized by the role of sulfur in the separation towards gender and Cu accumulation. Total sulfur, as determined by INAA via 37S, hardly shows any correlation with INAA Cu liver values (correlation coefficients 0.03 and 0.32 for nail and hair, respectively). However, the INAA results provide only the total sulfur content, i.e., sulfur present in keratin, melanin, and any other compound, whereas the PCs of the channel contents of the X-ray spectra, especially due to the peakless scattered region, apply to sulfur directly related to keratin and/or melanin. This explains the much better correlation of “keratin/melanin” sulfur with Cu liver values compared with total S with Cu.
The results of our study show that a fairly sharp differentiation can be obtained between dogs with low and high Cu liver levels if PCA is performed on a dataset obtained from all channel contents of X-ray spectra obtained by X-ray tube excitation of nail or hair samples together with the spectral information of often neglected X-ray (Rayleigh, Raman) scattered radiation.
A practical application of this technique would start with construction of a dataset using samples of known origin, encompassing all varieties expected. New and unknown samples can then be projected on this model, and subsequently classified. This approach will be further validated with new samples to be collected from Labrador Retrievers still being observed and treated along medicinal and dietary approaches.
Conclusions
Hair and nail clippings are widely used as bioindicators for the trace element status of man and, to a much lesser extent, also for animals. The results of this study emphasize that it is quite risky to assume a priori that the trace element levels in hair and nail reflect differences between classes of individuals, e.g., “healthy” and “sick” people or animals. In this study related to chronic hepatitis, the Cu levels in liver biopsies are not reflected by Cu levels in hair and nail.
The X-ray Rayleigh scattered and X-ray Raman continuum contain valuable information about the organic, low-Z elemental matrix composition of an object. In this study, first indications were obtained that the Cu accumulation possibly may have an effect on the nail and hair melanin, thus offering a potentially favorable outlook for noninvasive monitoring of the Cu liver status. An additional advantage of the approach is that a measurement by X-ray tube excitation takes only about 2 min per sample so that, once a database has been built up, the technique might be valuable for large-scale screening purposes.

neutron activation analysis
trace elements
chemometrics
x-ray spectroscopy
biological samples


Risk of Fecal Diversion in Complicated Perianal Crohn’s Disease

The purpose of the study was to determine the overall risk of a permanent stoma in patients with complicated perianal Crohn’s disease, and to identify risk factors predicting stoma carriage. A total of 102 consecutive patients presented with the first manifestation of complicated perianal Crohn’s disease in our outpatient department between 1992 and 1995. Ninety-seven patients (95%) could be followed up at a median of 16 years after first diagnosis of Crohn’s disease. Patients were sent a standardized questionnaire and patient charts were reviewed with respect to the recurrence of perianal abscesses or fistulas and surgical treatment, including fecal diversion. Factors predictive of permanent stoma carriage were determined by univariate and multivariate analysis. Thirty of 97 patients (31%) with complicated perianal Crohn’s disease eventually required a permanent stoma. The median time from first diagnosis of Crohn’s disease to permanent fecal diversion was 8.5 years (range 0–23 years). Temporary fecal diversion became necessary in 51 of 97 patients (53%), but could be successfully removed in 24 of 51 patients (47%). Increased rates of permanent fecal diversion were observed in 54% of patients with complex perianal fistulas and in 54% of patients with rectovaginal fistulas, as well as in patients that had undergone subtotal colon resection (60%), left-sided colon resection (83%), or rectal resection (92%). An increased risk for permanent stoma carriage was identified by multivariate analysis for complex perianal fistulas (odds ratio [OR] 5; 95% confidence interval [CI] 2–18), temporary fecal diversion (OR 8; 95% CI 2–35), fecal incontinence (OR 21, 95% CI 3–165), or rectal resection (OR 30; 95% CI 3–179). Local drainage, setons, and temporary stoma for deep and complicated fistulas in Crohn’s disease, followed by a rectal advancement flap, may result in closing of the stoma in 47% of the time. The risk of permanent fecal diversion was substantial in patients with complicated perianal Crohn’s disease, with patients requiring a colorectal resection or suffering from fecal incontinence carrying a particularly high risk for permanent fecal diversion. In contrast, patients with perianal Crohn’s disease who required surgery for small bowel disease or a segmental colon resection carried no risk of a permanent stoma.
Introduction
Crohn’s disease was initially described as a nonspecific inflammatory bowel disease, affecting mainly the terminal ileum and characterized by a subacute or chronic necrotizing and cicatrizing inflammation.1 Eventually, gastrointestinal Crohn’s disease became recognized as a full-thickness disease of the gastrointestinal wall that may affect the entire gastrointestinal tract, including the perianal region.2–6
Perianal lesions are common in patients with Crohn’s disease.7–13 Clinical manifestations vary from asymptomatic skin tags to severe, debilitating perianal destruction and sepsis. Asymptomatic perianal lesions require no treatment, but because they become painful and disabling, they may require surgical treatment. Surgical management needs to be conservative and should focus on the drainage of septic sites, preserving sphincter function and palliating symptoms.10–13 Medical management has had some success in improving symptoms, but as yet, it has not been able to eliminate most perianal complaints permanently.14,15
Fecal diversion was successfully used to achieve remission in colonic Crohn’s disease. Moreover, it was utilized to allow severe perianal disease to settle, thereby avoiding proctectomy.16–19 However, restoring the intestinal passage carries the risk of recurrent perianal disease activity, possibly resulting in a decreased quality of life compared to the situation with fecal diversion.
We investigated the overall risk of a permanent stoma in patients with severe perianal Crohn’s disease and tried to identify risk factors predicting permanent stoma carriage.
Methods
Patients
A total of 102 consecutive patients with Crohn’s disease presented with the first manifestation of a perianal fistula or a perianal abscess in our outpatient department between 1992 and 1995. Patients were investigated in Trendelenburg’s position by perianal inspection, proctoscopy, rectoscopy, and rigid sigmoidoscopy. Endoanal ultrasound was performed in case of suspected perianal abscess formation, and MRI was conducted of the pelvic floor in case of complicated fistulizing disease or intrapelvic abscess formation. All patients were documented prospectively. Follow-up data of 97 patients (95%) were available by a standardized questionnaire mailed to the patients and by a standardized chart review. There were 50 female and 47 male patients (ratio 1.06:1) with a median age of 23 years (range 8–51 years). Patients were evaluated with respect to the recurrence of perianal abscesses, fistulas, or surgical treatment of Crohn’s disease over the years. The median interval between the first diagnosis of Crohn’s disease and last follow-up was 16 years (range 8–37 years). Four patients had isolated small intestinal disease, 11 patients had isolated colonic disease, and 82 patients had small intestinal and colonic disease.
The abscess location was categorized as subcutaneous, intersphincteric, deep perianal, ischiorectal, and above the pelvic floor. Abscess formations were categorized into simple (subcutaneous, intersphincteric, deep perianal, and ischiorectal, circular extension less than 90°, pelvic floor not involved) and complicated (circular extension more than 90° [horse shoe abscess] or pelvic floor involved). Fistulas were classified according to Parks et al.20 into subcutaneous, intersphincteric, extrasphincteric, transsphincteric, rectovaginal, and suprasphincteric, as described previously.21 We divided fistulas into simple fistulas (no more than two perianal openings) and complex fistulas (rectovaginal, three or more perianal openings).
A variety of factors, such as sex of the patient, perianal fistula, rectovaginal fistula, abscess formation, anal stricture, fecal incontinence, or abdominal surgery were evaluated with regard to their predictive character for permanent stoma carriage by univariate and multivariate analysis.
Statistical Analysis
Kaplan–Meier analysis using a log-rank test was used for comparing risk rates over time. Factors that might influence permanent fecal diversion were analyzed using the chi-square test. Fisher’s exact test was used for univariate analysis and multiple logistic regression and Wald’s test for multivariate analysis. Subgroup analysis was performed for rectovaginal fistulas, as present in female patients only. Data are given as numbers of cases and percentages or median and interquartile ranges. A P < 0.05 was considered as significant.
Results
Overall Risk of Permanent Fecal Diversion
Thirty of 97 patients (31%) with perianal Crohn’s disease eventually required a permanent stoma. Nineteen patients were female and 11 male (ratio 1.73:1). The median time from first diagnosis of Crohn’s disease to permanent fecal diversion was 8.5 years (range 0–23 years; Fig. 1). Increased rates of permanent fecal diversion were observed in 53% of patients with previous temporary fecal diversion, in 54% of patients with complex perianal fistulas, in 54% of female patients with rectovaginal fistulas, and in 83% of patients with fecal incontinence (Table 1). Patients who required subtotal colon resection (60%), left-sided colon resection (83%), or rectal resection (93%) needed a permanent stoma at high rates (Table 2).
Figure 1Relative proportion of patients without permanent stoma in complicated perianal Crohn’s disease (n = 97) during follow-up.Table 1Patients with First Manifestation of Complicated Perianal Crohn’s Disease (n = 97) and the Rate of Fecal Diversion During Follow-up PatientsTemporary StomaPermanent StomaOverall9751 (53%)30 (31%)Abscess  formation7532 (43%)21 (28%)Simple abscess formation33 (44%)13 (39%)7 (21%)Complex abscess formation42 (56%)23 (55%)15 (36%)Fistulas8851 (58%)26 (30%)Simple perianal fistulas42 (48%)17 (40%)8 (19%)Complex perianal fistulas46 (52%)34 (74%)23 (50%)Rectovaginal fistulas26 (54% of ♀)18 (69% of 26)14 (54% of 26)Overlap between abscess formation and presence of fistulas exists, and abscesses led to fecal diversion in combination with fistulas only.Table 2Abdominal Procedures and the Rate of Fecal Diversion During Follow-up in Patients with Complicated Perianal Crohn’s Disease (n = 97) Patient NumberPermanent Stoma (% of Patients)Small Bowel ProceduresStricturplasty70Small bowel resection because of stenosis230Small bowel resection because of enteroenteric fistula70Anastomosis resection because of inflammatorystenosis300Stoma revision40Large bowel proceduresSegmental colon resection140Right-sided colon resection204 (20)Ileocecal resection6824 (35)Subtotal colon resection3521 (60)Left-sided colon resection65 (83)Rectal resection1312 (92)Patients that required small bowel resections carried no risk of fecal diversion, whereas patients with colon resections carried an increased risk of fecal diversion.
Abscess Formation
Seventy-five of 97 patients (77%) had at least one perianal abscess at first presentation or during follow-up. Surgical therapy for abscesses consisted of seton drainage, mushroom catheter drainage, or incision and drainage, as described previously.21 A permanent fecal diversion because of recurrent abscess formations, always in combination with fistula problems and perianal sepsis, became necessary in 21 of 75 cases (28%). Simple abscess formations occurred in 33 patients, and 7 of those patients (21%) required permanent fecal diversion (Fig. 2). Complex abscess formations were present in 42 patients, with permanent fecal diversion being necessary in 14 cases (33%; Table 1).
Figure 2Relative proportion of patients without permanent stoma in complicated perianal Crohn’s disease with simple (n = 42) and complex (n = 46) fistulas during follow-up. The presence of a complex fistula significantly increased the risk of permanent fecal diversion (P < 0.001).
Perianal and Rectovaginal Fistulas
Perianal fistulas affected 88 of 97 patients (91%), including 26 female patients with rectovaginal fistulas. Symptomatic fistulas with abscess formation were treated by simple drainage procedures and provided with a seton. Azathioprine therapy was tried if purulent secretion persisted. Infliximab was rarely used in recent years at the discretion of the referring gastroenterologist. Overall, permanent fecal diversion became necessary in 26 of 88 patients (30%). Forty-two patients (48%) had simple fistulas, whereas 46 patients (52%) had complex fistulas. Of these, permanent fecal diversion was documented in 14 patients (54%), whereas simple fistulas eventually required a permanent stoma in only 8 cases (16%). Twenty-six of 48 female patients had rectovaginal fistulas, of which 14 (54%) eventually required permanent fecal diversion (Table 1).
Thirty-four of 97 patients (35%) developed anal strictures, whereas fecal incontinence was documented in 12 patients (12%) during the follow-up. In 14 of 34 patients (41%) with anal stricture, a permanent fecal diversion became necessary, whereas 10 of 12 patients (83%) suffering from fecal incontinence required a permanent stoma.
Abdominal Surgery
Eighty-three of 97 patients with perianal Crohn’s disease (86%) underwent abdominal surgery at least once during follow-up because of Crohn’s disease activity, with a total of 227 abdominal procedures being performed. In 68 of 227 operations (29%), intestinal stenosis required surgery. Segmental colon resection was performed 108 times (46%), whereas subtotal colectomy for fulminant colitis was performed 35 times (15%). One patient required surgery because of a fistula carcinoma. Patients with complicated perianal Crohn’s disease who required a small bowel resection carried no risk of permanent fecal diversion, whereas the majority of patients with left-sided colon resection, subtotal colon resection, or rectal resection needed permanent fecal diversion (Table 2). Patients that needed abdominal surgery repeatedly carried an increased risk for a permanent stoma (permanent stoma rate: three or more abdominal operations, n = 20, 50%; less than two abdominal operations, n = 20, 15%; P < 0.043). Patients with permanent fecal diversion had undergone previous abdominal surgery three times as often as those patients without (P < 0.05; Table 3). Eventually, 17 patients (18%) needed proctectomy, of which 13 were female patients with rectovaginal fistulas. In 14 patients with a permanent stoma, a rectal stump remained in place and was controlled at yearly intervals. Patients with complicated perianal Crohn’s disease and large bowel resection carried a significantly increased risk of permanent fecal diversion compared to patients with complicated perianal Crohn’s disease and small bowel resection (48 vs 0%, P < 0.001) (Fig. 3).
Table 3Abdominal Procedures per Patient and Percentage of Patients with Abdominal SurgeryAbdominal ProceduresMedian(25–75%)Patients with Abdominal Surgery (%)P ValuePermanent fecal diversion3(2–4)100P < 0.05Without permanent fecal diversion1(1–3)Temporary fecal diversion3(2–4.25)80P < 0.05Without temporary fecal diversion1(0–2.5)Anal stricture2(1–4)60n.s.Without anal stricture2(1–3)Incontinence2(2–4.5)10n.s.Without incontinence2(1–3)Patients with Crohn’s disease and complicated perianal fistulas who required temporary or permanent stoma had more abdominal procedures than patients who did not need a stoma, whereas patients with anal stricture or fecal incontinence had no increased abdominal procedure rate (the number of abdominal procedures per patient is given as median with interquartile ranges). Abdominal surgery and fecal diversion correlated on univariate analysis (P < 0.05).Figure 3Treatment algorithm for patients with complicated perianal Crohn’s disease.
Temporary Fecal Diversion
Temporary fecal diversion, done mostly by loop ileostomy, became necessary in 51 of 97 patients (53%). Increased rates of temporary fecal diversion were observed in 55% of patients with complex abscess formations, including those with severe perianal sepsis, in 77% of patients with complex perianal fistulas, and in 69% of female patients with rectovaginal fistulas. In 24 of 51 patients (47%) the temporary stoma could be removed after perianal disease had subsided. Fistulas were closed in these patients by rectal advancement flaps,23 whereas cutting setons, fistulectomy, or infliximab infusions were not employed in these patients.
Risk Factors Predictive of a Permanent Fecal Diversion
Univariate Analysis
Complex perianal fistulas (P < 0.04), fecal incontinence (P < 0.001), and rectovaginal fistulas in female patients (P < 0.001) carried an increased risk for a permanent stoma. Patients with high rates of abdominal surgery had a significantly increased risk for fecal diversion, whereas the frequency of abdominal surgery was not influenced by the presence of anal stricture or fecal incontinence (Table 3). The need for subtotal colon resection (P < 0.001), rectal resection (P < 0.001), or temporary fecal diversion (P = 0.001) also resulted in an increased risk of permanent fecal diversion. A variety of factors did not increase the risk of a permanent stoma, such as sex of the patient, anal stenosis, perianal abscesses, and abdominal surgery, excluding subtotal colon and rectal resection (Table 4).
Table 4Risk Factors for Permanent Fecal Diversion by Univariate and Multivariate Analysis in Patients with Complicated Perianal Crohn’s DiseaseRisk FactorsUnivariate AnalysisMultivariate AnalysisPermanent Fecal Diversion (%)P ValueOR95% CIP ValueRectal resection92P < 0.001305–179P < 0.002Fecal incontinence83P < 0.001213–165P < 0.02Subtotal colectomy60P < 0.001Rectovaginal fistulas54P < 0.001Temporary fecal diversion51p = 0.00182–35P < 0.02Complex perianal fistulas54P < 0.0452–18P < 0.03
Multivariate Analysis
According to multivariate analysis, complex perianal fistulas (P < 0.03), fecal incontinence (P < 0.02), temporary fecal diversion (P < 0.02), or rectal resection (P < 0.002) still carried a significantly increased risk for permanent fecal diversion (Table 4).
Discussion
Perianal disease is a feature of Crohn’s disease that afflicts approximately one third of patients, but prevalence rates vary between 8 and 90%.19 Patients with colonic involvement will have perianal disease in more than 50% of cases, whereas patients with small bowel disease are affected in less than 20%.24 Perianal Crohn’s disease can present as minor lesions, such as skin tags, skin excoriations, and fissures, which rarely need surgical treatment. In contrast, perianal fistulas often result in abscesses or perianal sepsis, requiring urgent surgery, and fistulas and abscesses are considered as suppurative complications of perianal Crohn’s disease.25 We conducted surgery as conservatively as possible because sphincter function should be preserved as much as possible.19,22,23 Throughout the study period, 77% of patients developed abscess formations, which is considerably more than the 48% rate reported previously with a shorter mean follow-up of 32 months.22
The incidence of perianal fistulas in Crohn’s disease is somewhat lower, affecting 10–34% of patients.26 Healing rates of 60–70% in patients with Crohn’s disease and perianal fistulas were reported in earlier series.21,26–28 However, these series contained a large proportion of simple fistulas, whereas most of our patients had complex transsphincteric or rectovaginal fistulas and fistulas that did not heal through conservative treatment. Perianal fistulas that resulted in abscess formation were drained surgically, and in case of massive purulent secretion or perianal sepsis, oral antibiotics were used additionally. Setons were put into place to prevent recurrent abscess formation, a treatment that is well known as a possible means for controlling perianal sepsis and preventing recurrent abscess formation.19,21–23,29 We did not use cutting setons nor fistulotomy for transsphincteric fistulas, as fecal incontinence may succeed.19,22,23 Infliximab was successfully shown to reduce fistula secretion, and outer fistula openings may heal.30 However, fistula tracks persist with varying degrees of residual inflammation, which may cause recurrent fistulas and perianal abscesses.31
Rectal advancement flaps can be used to close the inner opening of transsphincteric or rectovaginal fistulas when perianal disease has abated and the rectal mucosa is not inflamed. However, fistulas eventually recurred in about one third of patients after a mean follow-up of 19 months, and some patients developed new fistulas.23 Hyman32 reported an initial 79% healing rate in a series of 14 patients with Crohn’s disease and perianal fistulas, but observed longer term success in only 50% of patients. Our current approach is to use rectal advancement flaps for transsphincteric fistulas while delaying surgery if there is an acute flare of Crohn’s disease, in particular, if proctitis is present.23,33 Perianal fistulas in Crohn’s disease closed by a rectal advancement flap have a recurrence rate of 50–60%, but can be attempted repeatedly.23,34 If a second rectal advancement flap fails, the failure rate increases up to 75%,34 and a temporary stoma is suggested to the patient before a third attempt is made. By this approach, we were able to heal perianal fistulas in 24 out of 51 patients (47%) who required a temporary stoma. In a previous study, diversion was the most powerful factor influencing healing according to multiple regression analysis.23 The chances of a symptomatic fistula recurrence were increased without a stoma (52% vs 14%, 21), and patients with Crohn’s colitis carried an increased risk of fistula recurrence.23
A particular problem is rectovaginal fistulas, which almost always open at the dentate line.19 They occur in 3–10% of patients with Crohn’s disease.19 In our series, 54% of female patients had rectovaginal fistulas, indicating that a large patient proportion had complex perianal fistulas; referral bias might have contributed to this high proportion of rectovaginal fistulas, as our outpatient clinic for Crohn’s disease is well known regionally. Rectovaginal fistulas carry a poor prognosis,18 and a 70% recurrence rate was observed 24 months after a rectal advancement flap was performed.23 In the experience of Keighley et al.35, 11 of 13 patients with rectovaginal fistulas required proctectomy with a permanent stoma. In our series, 8 out of 26 rectovaginal fistulas healed by a rectal advancement flap. Eighteen patients required a temporary stoma, of which four could be removed after a rectal advancement flap was tried again successfully (overall healing rate 12 of 26, 46%), resulting in a permanent stoma rate of 14 of 26 patients (54%). Healing rates of rectovaginal fistulas associated with Crohn’s disease vary widely, and low patient numbers are usually reported.18 In the Cleveland clinic, 16 of 37 rectovaginal fistulas (43%) healed using an endorectal advancement flap, but patients not having Crohn’s disease were included in the study.36 In low anovaginal fistulas, higher healing rates of up to 68% were reported in patients with Crohn’s disease.37 Recurrent rectovaginal fistulas were shown to heal after repeated rectal advancement flap procedures were conducted at about the same rate even in Crohn’s disease, but surgery should be delayed for at least 3 months after a previous repair,38 which is standard policy at our clinic. It is interesting to note that Halverson et al.38 reported that the presence of a diverting stoma significantly increased the fistula recurrence rate (67 vs 50%).
After years of perianal or transvaginal pus secretion and recurrent abscess formations, patients are often tired of the suffering involved. Antibiotics, azathioprine, or 6-mercaptopurine may be used successfully in suppurative perianal Crohn’s disease.15,18,38,39 In our department, this is routinely tried if perianal disease does not settle after surgical drainage is provided. However, azathioprine or 6-mercaptopurine are not tolerated by all patients and may also fail to relieve perianal Crohn’s disease. In these cases, fecal diversion can be offered as a relatively minor procedure with low morbidity,18 and might be performed laparoscopically in selected cases.40 However, overall healing rates of perianal disease are only around 40%, with 87% of those being diverted eventually retaining their stoma.18 We diverted 51 patients, of which 27 retained there stoma (53%), which compares favorably, but indicates a considerable risk for a permanent stoma if diversion is tried, and being a significant risk factor according to multivariate analysis in our series.
Perianal disease in combination with fecal incontinence might necessitate fecal diversion as well. Factors that may contribute to fecal incontinence in patients with Crohn’s disease are reduced stool consistency, sphincter injuries after abscess incisions, and keyhole deformities after laying open perianal fistulas. The latter was reported to result in fecal incontinence in 5 of 27 patients with perianal fistulas in Crohn’s disease,41 which is why we did not use this technique or cutting setons in transsphincteric fistulas. We always incise perianal abscesses along a circular perianal line, meticulously preserving sphincter integrity. Nevertheless, fecal incontinence became prevalent in 12% of our patients during follow-up and was a significant predictor of permanent fecal diversion by multivariate analysis. Rectal advancement flap procedures may influence continence,22 and a 9% rate of worsened continence was reported thereafter.33 In particular, the risk of fecal incontinence was increased after flap repairs in patients with previous surgical fistula repairs,33 but only 2 out of 12 patients with fecal incontinence had a previous rectal advancement flap repair in our study. A high rate of abdominal procedures was associated with an increased risk for a temporary or a permanent stoma, possibly reflecting high intestinal disease activity of Crohn’s disease in these patients. All 30 patients that ended with a permanent stoma had frequently undergone abdominal procedures (median of three procedures).
Because of the suffering involved and unsuccessful previous attempts to heal perianal fistulas, about half of our patients eventually opted for temporary ileostomy, being brought into contact with a stoma nurse and stoma carriers with Crohn’s disease before, and 31% of all patients remained with a permanent stoma. In general, the need for a stoma was reduced during recent decades in Crohn’s disease,43 but the overall long-term risk for a stoma in patients with Crohn’s disease who require abdominal surgery was reported between 30 and 40%.44,45 Fecal diversion remains an option to subside perianal disease activity, with an early response rate of about 70–80%.46–48 Unfortunately, 75% of those patients eventually experienced a relapse, and the restoration of intestinal continuity was achieved in 10% only.48 In 130 patients with surgically treated perianal Crohn’s disease, a permanent stoma became necessary in 24%, and this rate increased to 53% in patients with Crohn’s colitis.45 In this light, considering that all of our patients had complicated perianal disease and almost all (93 of 97 patients, 96%) had colonic involvement, we judge a 31% permanent stoma rate as a success in a subset of patients carrying a particularly high risk for a stoma. Local drainage, setons, and, if not successful, a temporary stoma after a rectal advancement flap, resulted in closure of the stoma in 47% of the time, which is an improvement compared to previous studies where restoration of intestinal continuity was reported in only 10–40% of patients, with most studies providing the lower end percentages.44,46–48 If perianal Crohn’s disease was the indication for creating a stoma, stoma closure was successful in 2 out of 15 patients only (13%).47
Eventually, proctectomy became necessary in 18% of patients, which is in the range others have reported.24,44,49,50 However, if the presence of colonic disease was considered separately, seven out of eight patients with complicated perianal Crohn’s disease needed proctectomy, again indicating that an 18% proctectomy rate in our high-risk population is rather low. All of our patients who needed proctectomy had colonic disease, and 13 of 17 had a rectovaginal fistula.
In a recent study investigating the quality of life in patients with Crohn’s proctocolitis, patients in remission had a health-related quality of life similar to controls. Patients with active disease had a reduced quality of life, and only the symptom index negatively predicted a reduced quality of life, whereas neither previous colonic surgery nor the presence of a stoma did.51 Accordingly, carrying a stoma does not necessarily mean a reduced quality of life, although a slight reduction in two out of eight domains (physical and emotional role) was detected.51 However, in patients with perianal disease, the cumulative abscess rate during 3 years of follow-up and the symptomatic fistula recurrence rate with a mean follow-up of 22 months were both greatly reduced with a stoma (76 vs 13% and 52 vs 14%), indicating reduced suffering and need for surgery.21,22
In summary, patients with complicated perianal Crohn’s disease, colonic involvement, and a high rate of abdominal procedures carried a significant risk for a permanent stoma. However, if the risk factors identified were taken into account, the rate of patients eventually requiring a permanent stoma seemed low and showed a decrease compared to previous studies, indicating that multiple treatment episodes and complex surgery, including temporary fecal diversion, might eventually heal at least some patients in this high-risk population, as was also observed by others.50

fecal diversion
crohn’s disease
perianal abscesses
fistulas