[Management Strategy and Evaluation of Surgical Outcomes in Patients with Recurrent Patellar Instability between 2010-2020]. / Strategie lécby a vyhodnocení výsledku operací recidivujících patelárních nestabilit mezi roky 2010­2020.

PURPOSE OF THE STUDY The study retrospectively reviews the outcomes of patella stabilisation surgeries performed at our department in the period 2010-2020. It aimed to provide a more thorough evaluation, to compare the respective types of MPFL reconstruction and to confirm the beneficial effect of tibial tubercle ventromedialization on patella height. MATERIAL AND METHODS In the period 2010-2020, a total of 72 stabilisation surgeries of patellofemoral joint in 60 patients with objective patellar instability (OPI) were performed at our department. The surgical treatment outcomes were evaluated retrospectively using a questionnaire, including the postoperative Kujala score. A comprehensive examination was carried out in 42 patients (70%) who had completed the questionnaire. In case of distal realignment, the TT-TG distance and a change in the InsallSalvati index which serve as an indication for surgery, were assessed. RESULTS Altogether 42 patients (70%) and 46 surgical interventions (64%) were evaluated. The follow-up period was 1-11 years, with the mean follow-up of 6.9 years. In the studied group of patients, only 1 case (2%) of new dislocation was seen, in 2 cases (4%) the patients reported a subluxation episode. The mean score using the school grades was 1.76. Thirty-eight patients (90%) were satisfied with the surgical outcome, 39 patients would undergo a surgery in case of identical problems with the other limb. The mean postoperative Kujala score was 76.8 points, range 28-100 points. The mean TT-TG distance in the studied group with the preoperative CT scan (33x) was 15.4 mm (12-30 mm). The mean TT-TG distance in the cases indicated for tibial tubercle transposition was 22.2 mm (15-30 mm). The mean Insall-Salvati index prior to the performance of tibial tubercle ventromedialization was 1.33 (1-1.74). Postoperatively, the index decreased by 0.11 on average (-0.00 to -0.26) to 1.22 (0.92-1.63). No infectious complications were presented in the studied group. DISCUSSION In patients with recurrent patellar dislocation, the instability is often times caused by pathomorphologic anomalies of the patellofemoral joint. In patients with clinically expressed patellar instability and physiological values of the TT-TG distance, an isolated proximal realignment is performed by medial patellofemoral ligament (MPFL) reconstruction. In the case of pathological values of the TT-TG distance, distal realignment is performed by tibial tubercle ventromedialization to achieve physiological values of the TT-TG distance. In the studied group, tibial tubercle ventromedialization helped decrease the Insall-Salvati index by 0.11 points on average. This has a positive side effect on the patella height, thus on increasing its stability in the femoral groove. In patients with both proximal and distal malalignment, a two-stage surgery is performed. In the isolated cases of severe instability or if symptoms of lateral patellar hyperpressure are present, musculus vastus medialis transfer or arthroscopic lateral release are performed as well. CONCLUSIONS When correctly indicated, proximal, distal realignment or their combination can bring very good functional outcomes with a low risk of recurrent dislocation and postoperative complications. The importance of MPFL reconstruction is confirmed by low incidence of recurrent dislocation in the group investigated in this study, namely when compared with studies referred to in this paper, in which the patients underwent patellar stabilisation using the Elmslie-Trillat procedure. Conversely, leaving the bone malalignment untreated during the isolated MPFL reconstruction increases the risk of its failure. Judging from the obtained results, tibial tubercle ventromedialization also has a positive effect on the patella height through its distalization. Provided the stabilisation procedure is correctly indicated and performed, the patients can get back to their normal activities, often even sports activities. Key words: objective patellar instability, patellar stabilisation, MPFL, tibial tubercle transposition.

[Accuracy of Alignment of Femoral and Tibial Component of the Oxford Medial Unicompartmental Knee Arthroplasty Using Zimmer Microplasty® Instrumentation]. / Presnost centrace femorální a tibiální komponenty mediální unikompartmentální náhrady kolenního kloubu Oxford za pouzití instrumentária Zimmer Microplasty®.

PURPOSE OF THE STUDY The purpose of the study is to verify the correct alignment of components of the Oxford medial unicompartmental knee arthroplasty using the Zimmer Microplasty® instrumentation at the beginning of the learning curve. The implantation of prosthetic components of partial knee arthroplasty in proper alignment has an effect on long-term survival of the prosthesis and should eliminate the occurrence of frequent complications. MATERIAL AND METHODS The study group includes 20 patients, 9 men with the mean age of 68 years (range 62-78 years) and 11 women with the mean age of 69 years (range 52-81 years). 13 patients underwent surgery on the right knee and 7 patients on the left knee. The mean length of symptoms was 13 months (range 7-20 months), the mean varus knee deformity was 7° (range 4-12°). The selected patients met both the clinical and radiological criteria for enrolment in the study. In all of them medial unicompartmental knee arthroplasty was performed in 2019 by the same surgeon. After surgery, the parameters of accuracy of alignment of individual prosthetic components were measured on the full-length radiograph of the limb under load. The WOMAC score was recorded preoperatively and one year after surgery and subsequently compared. The knee joint flexion preoperatively and one year after surgery was evaluated. RESULTS The analysis of radiographs revealed that in 12 cases the femoral component was implanted in the mean valgus angle of 1.6° (range 1-3°) and in 8 cases in the mean varus angle of 3° (range 1-5°). All femoral components were implanted in the mean flexion of 7.3° (range 3-11°), no component was implanted in extension. As concerns the tibial component, 19 components were implanted in a neutral or minimum varus angle with the mean value of 1.1° (range 0.3-4°). One component only was implanted in the valgus angle of 1°. All tibial components were implanted with the mean dorsal slope of 6.5° (range 4-8°). The incision was 5 mm deep on average (range 3-6 mm). There was a slight divergence between the components, namely 2.8° on average (range 2-7°) and the distance between the components was 4mm on average (range 3-5 mm). The mean knee joint flexion achieved preoperatively by patients was 115° (range 110-123°), whereas postoperatively the mean flexion achieved was 126° (range 111-138°). The preoperative Womac score was 84.5 points on average (range 64-96 points), whereas postoperatively it was 26.4 points on average (range 7-52 points). None of the components was implanted outside the permitted range, no early complications of the partial replacement (luxation of polyethylene mobile bearing insert, early loosening of the prosthesis, tibial fracture) were observed. DISCUSSION Our radiographic measurements show that when Zimmer Microplasty® instrumentation is used correct alignment of the femoral and tibial component can be achieved and the individual components were correctly aligned within the recommended range. Every single component met the required criteria for alignment. When comparing the values obtained by us in measurements with those obtained by other authors from abroad, similar results regarding the alignment of components were achieved. CONCLUSIONS When using Zimmer Microplasty® instrumentation, excellent results can be achieved also at the beginning of the learning curve of partial knee replacement. Key words: medial gonarthrosis, unicompartmental arthroplasty, hemiarthroplasty, alignment of components.

[Native Joint Septic Arthritis in Adults: Incidence in Our Group of Patients and Antibiotic Therapy Guidelines]. / Infekcní artritidy nativních kloubu u dospelých: výskyt v nasem souboru pacientu a doporucení antibiotické lécby.

PURPOSE OF THE STUDY The purpose of the study is to analyse the number of adult patients treated in our department for native joint septic arthritis and to outline guidelines for antibiotic therapy. MATERIAL AND METHODS From the beginning of 2003 to the end of 2020, a total of 36,342 surgeries were performed at our department. We retrospectively reviewed and analysed all surgeries for native joint septic arthritis (a total of 538 surgical interventions). The study included all adult patients who were operated for native joint septic arthritis in our department in 2003-2020. We included all revision surgeries for ongoing infection (excluding the management of post-infectious findings) as well as all operations performed in patients with multiple joint involvement. Based on the analysis of our data and review of published guidelines for antibiotic treatment of septic arthritis, we have outlined our own antibiotic therapy guidelines for the treatment of native joint septic arthritis. RESULTS From 2003 to 2020 we performed a total of 36,342 surgeries, of which 538 (1.5%) in 461 patients was indicated for native joint septic arthritis. The cohort consisted of 292 men (63%), who underwent 344 surgeries, and 169 women, in whom 194 surgeries were performed. The mean age of patients irrespective of the arthritis location was 62.4 years. Altogether, 19 patients (4.1%) suffered from multi-joint arthritis. The most frequently operated joint was the knee with 252 (54%) patients and 300 surgeries (56%), followed by the shoulder with 68 (14.7%) patients and 78 (14.5%) surgeries, the hip with 38 (8.2%) patients and 42 surgeries (8%), the carpal with 30 (6.5%) patients and 35 (6.5%) surgeries, the ankle with 25 patients (5.4%) - 31 (6%) surgeries, the small finger joints with 22 (4.75%) patients and 23 (4%) surgeries, the elbow with 14 (3%) patients and 14 (2.6%) surgeries, the sternoclavicular joint with 9 (1.9%) patients and 12 (2.2%) surgeries and the acromioclavicular joint with 3 patients and 3 (0.5 %) surgeries, respectively. DISCUSSION The management of septic arthritis relies heavily on early diagnosis, early surgical intervention and adequate antibiotic therapy. The diagnostic process and surgical treatment have their specifics related to the affected location, therefore, respective guidelines will be published separately for each location including the results. On the other side, antibiotic management is not dependent on the location and therefore the guidelines are included in this first analysis septic arthritis in the whole cohort. CONCLUSIONS Septic arthritis in adults in an ongoing issue with rising incidence. Early diagnosis, urgent and adequate surgical treatment, and optimal antibiotic therapy are preconditions for successful outcome. Key words: native joint septic arthritis, incidence, antibiotic therapy, guidelines.

[Outcomes of Revisions of the Acetabular Component of THA with Paprosky Type 3a and 3b Defects Using Tantalum Trabecular Metal Implants 2-10 Years Postoperatively]. / Výsledky revizí acetabulární komponenty TEP kycle s defekty Paprosky 3a a 3b pomocí trabecular metal tantalových implantátu 2­10 let po operaci.

PURPOSE OF THE STUDY Tantallum trabecular metal implants (Trabecular Metal Technology - TMT) considerably changed the acetabular reconstruction options in revision surgeries with extensive bone defects and distorted pelvic ring integrity. The purpose of this study is to ascertain the short-term to medium-term outcomes of acetabular reconstruction through TMT implants in patients with Paprosky type 3a and 3b acetabular defects and in case of pelvic discontinuity. MATERIAL AND METHODS The prospective monocentric study included patients in whom the revision of acetabular components in total hip arthroplasty was performed, the acetabular defect was classified as Paprosky 3a and higher, a TMT implant was used for reconstruction, and the follow-up period was at least 2 years after surgery. In total, 87 patients who had met the inclusion criteria were operated on and followed-up. The patients in the study group underwent a clinical examination, an X-ray and also an assessment using the Harris hip score. Moreover, the patients were asked about their satisfaction with the surgical outcome, their willingness to undergo the same procedure again in case of difficulties, and they were also asked to rate the outcome in percentage term and by assigning grades. Also, an analysis of the reasons for revision and subsequent complications was carried out. Implant integration and its migration were evaluated on an X-ray. RESULTS 32 men and 55 women were subjects to evaluation, with a balanced number of operated sides (44:43 in favour of the right side). One-stage procedures prevailed, which were performed in a total of 74 cases, while two-stage revisions were performed in 13 cases in the study group. In three patients (3.5%) pelvic discontinuity was diagnosed, 69 patients (79%) suffered from Paprosky 3a defect and 15 patients (17%) from Paprosky 3b defect. The first patients underwent surgery in 2009 and the mean follow-up period in the study group was 48 months. In 1 patient the TMT implant was removed for infectious complications, in the remaining part of the group the TMT implant was fully integrated with no signs of loosening or migration in the monitored period. In the assessment using the Harris hip score, the mean score of 80.4 (range 36-99) was achieved. When assessing the satisfaction with the surgical outcome, the mean value achieved was 94.4%, and the mean assigned grade was 1.26 (on a school grading scale). DISCUSSION Extensive bone defects and pelvic discontinuity represent an issue in revisions of the acetabular component in total hip arthroplasty. There are several options how to address these conditions. State-of-the-art TMT implants thanks to their shortterm and medium-term outcomes appear as one of the most beneficial option with a low failure rate both in our study group and in published papers. CONCLUSIONS Evaluation of this monocentric prospective study reveals encouraging short-term and medium-term outcomes of the use of TMT implants in managing extensive acetabular bone defects of Paprosky 3a and 3b type and supports their further introduction into practice at our department. Key words: total hip arthroplasty (THA), revision implantation, TMT implant.

Molecular dynamics study of the effect of the gamma-Abu insert on the conformational behavior of the glycopeptide dendrimers based on the oligolysine scaffold in N, N'-dimethylformamide.

Glycodendrimers bearing Tn (alpha-D-GalNAc-(1 --> O)-Ser/Thr), an identified tumor-associated carbohydrate antigen, hold promise in the post-surgery treatment of a variety of tumors such as metastatic breast cancer. We used molecular dynamics (MD) techniques to examine structural differences taking place during synthesis of two classes of tetravalent Multiple Antigen Glycopeptides (MAG) that differ only by the gamma-Abu insert in the structure of the oligolysine core. Each of the selected intermediates of the synthesis was modeled, subjected to the 2 ns run in N,N'-dimethylformamide (DMF) and geometrically characterized. We characterized: a) distances of free, or extended termini from the anchor, b) interatomic distances between free or substituted N termini, c) radius of gyration and d) spatial distribution of molecular density. A detailed conformational analysis of 16 glycodendrimers shows the distinct behavior of the inserted vs. non-inserted constructs already during the first steps of the modeled synthesis. It suggests that the character as well as the length of the insert has a major impact on the spatial characteristics and behavior of the dendritic molecules. The inserts can, in principle, increase a tendency of dendrimers to establish a high-density core, which is similar to the effect of a higher generation.

Affinity chromatography of porcine pepsin on different types of immobilized 3,5-diiodo-L-tyrosine.

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.

Potentiometric and spectroscopic study of copper (II) ion binding by 1,6-anhydro-derivatives of aminosugars.

Potentiometric and spectroscopic studies of complexes of Cu2+ with two 1,6-anhydro derivatives of aminosugars, 1,6-anhydro-2-deoxy-2N-methylamino-beta-D-mannopyranose and 1,6-anhydro-3,4-epimino-beta-D-altropyranose, showed that the epimino nitrogen donor binds Cu(II) ions, but its position prevents formation of stable chelate complexes. The other ligand is very effective in metal ion coordination, but contrary to the homologous glucosamine derivative, it does not form any dimeric species.

Synthesis of S-linked glucosaminyl-4-thiohex-2-enopyranosides via allylic SN2' substitution of a tosylhexenose.

Treatment of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-beta-D-glucopyranose with 1,6-anhydro-3,4-dideoxy-2-O-p-toluenesulfonyl-beta-D-erythro-hex-3-enopyranose gave 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1-->4)-1,6-anhydro-2,3-dideoxy-4-thio-beta-D-erythro-hex-2-enopyranose in 86% yield. Its 1,6-anhydride bond was cleaved with methanol to give a mixture of methyl glycosides (alpha/beta approximately 5:1), from which the alpha anomer was separated by crystallization and converted into its 6-acetate, 6-methanesulfonate, or deacetylated to obtain the corresponding free methyl thiodisaccharide. The structure of the new compounds was confirmed by 1H and 13C NMR spectra.

The use of lectins for determination of absolute configurations of small amounts of sugars eluted from chromatograms.

A simple procedure for the determination of the absolute configuration (i.e., assignment to the D- or L-enantiomeric series) of glucose, mannose, galactose, fucose, arabinose, and rhamnose is described, based on inhibition by these sugars of 125I-labeled lectin binding to the glycoconjugates immobilized on the wells of plastic microculture plates. The method works well with 10 to 100-micrograms amounts of the sugars isolated after paper chromatography of the glycoprotein or polysaccharide hydrolysates.

A novel method for glycoconjugate synthesis.

2-Chloroethyl 1-thio-beta-D-galactopyranoside and the corresponding 1-thio-beta-D-glucopyranoside have been found to be suitable glycosylating agents for the preparation of different types of glycoconjugates. Glycosylation of bovine serum albumin and chymotrypsin were chosen as examples of an application of the described compounds. The glycosylating agents can modify not only amino groups, but also alkyl hydroxyl and aryl hydroxyl groups, as was shown in experiments with model water soluble and water insoluble polyacrylamide copolymers.

The development of L2X2Ru=CHR olefin metathesis catalysts: an organometallic success story.

In recent years, the olefin metathesis reaction has attracted widespread attention as a versatile carbon-carbon bond-forming method. Many new applications have become possible because of major advances in catalyst design. State-of-the-art ruthenium catalysts are not only highly active but also compatible with most functional groups and easy to use. This Account traces the ideas and discoveries that were instrumental in the development of these catalysts, with particular emphasis on (PCy3)2Cl2Ru=CHPh and its derivatives. The discussion includes an analysis of trends in catalyst activity, a description of catalysts coordinated with N-heterocyclic carbene ligands, and an overview of ongoing work to improve the activity, stability, and selectivity of this family of L2X2Ru=CHR complexes.

Separation of alpha-amino acid enantiomers by reversed-phase high-performance liquid chromatography after derivatization with o-phthaldialdehyde and a sodium salt of 1-thio-beta-D-glucose.

A sensitive system for D,L-amino acid analysis has been developed, using fluorescence derivatization with o-phthaldialdehyde in the presence of sodium salt of 1-thio-beta-D-glucose. The reagents rapidly form fluorescent diastereoisomeric derivatives with primary amino acids. These derivatives are efficiently separated on a conventional reversed-phase column with an analysis time of 60 min. Simultaneous determination of enantiomers of various amino acids was achieved by a simple binary gradient elution with methanol in 0.05 M aqueous sodium acetate.

Some immunochemical and chemical aspects of S and R Shigella dysenteriae 1 endotoxins.

Endotoxins of S and R forms of Shigella dysenteriae 1 were prepared by NaCl-Na citrate extraction, purified by gel chromatography on Sephadex G 200 and on Sepharose 4B and subjected to immunochemical and chemical analysis. The toxins contained 25--30% of lipids, 40--50% of carbohydrates and 14--24% of protein. The lipid and protein moieties of the lipopolysaccharide-protein complexes exhibited no significant difference, whereas the sugar moieties differed markedly (both qualitatively and quantitatively), in relation to the growth form of the culture. The lipid moiety, which consists at least of 22 fatty acids, has the greatest relative content (approx. 50%) of behenic acid, 22:0, and palmitic acid, 16:0 (approx. 11%). In the protein moiety, at least 16 amino acids were determined; these amino acids were identical in both endotoxin types, but their total content was higher in the R form, giving an R:S ratio of 1.7 +/- 0.2. The sugar moiety consists of galactose, glucosamine and either rhamnose (in S endotoxin) or aldoheptose (in R endotoxin). The difference of the chemical composition of the sugar moiety is believed to account for the diametric difference in the immunochemical character, in particular the different behaviour in the electric field, of both endotoxin types. The average content of 3-deoxy-D-manno-2-octulosonic acid was determined as 0.5% for both S and R endotoxin. Trace amounts of O-phosphorylethanolamine were found. Individual aspects of the chemical and immunochemical analysis are discussed in detail.

New trends in the use of Al(OH)3-conjugated endotoxins and their subunits from the S- and R-forms of Shigella dysenteriae serovar 1 for model vaccination purposes.

The lipopolysaccharides, lipopolysaccharide-protein complexes and their lipids A, isolated from Shigella dysenteriae 1, exhibited lethal toxicity (LD50 300-400 micrograms per mouse), pyrogenicity (0.01-1.0 microgram), activity in the Limulus test (10(-3)-10(-12) mg ml-1) and produced a positive local Shwartzman reaction. It was found that these substances bound to Al(OH)3 were capable of successfully protecting mice from challenge with both homologous and heterologous infections (up to 90% of mice survived a challenge with LD100), whereas immunization with substances with no biological carrier exhibited very low or zero protective effects. Furthermore, the positive cross-reactivity of lipids A in the agar immunodiffusion test confirms the wide applicability for vaccination purposes.

Selective N-debenzylation of benzylamino derivatives of 1, 6-anhydro-beta-D-hexopyranoses

When the series of 2-, 3-, and 4-(benzylamino)-2-, 3-, and 4-deoxy derivatives of 1,6-anhydro-beta-D-hexopyranoses in the D-gluco, D-lyxo, and D-arabino configurations were reacted with diisopropyl azodicarboxylate, N-benzyl groups were selectively cleaved in the presence of O-benzyl groups. The yields ranged from 51 to 97%. The debenzylation of some aliphatic benzylamines is also discussed.

Composition of lipid A in the S and R forms of Shigella dysenteriae serovar 1.

A chemical analysis was performed of lipid A, isolated by acid hydrolysis of the lipopolysaccharide from the S and R forms of Shigella dysenteriae serovar 1. Differences in the moiety of both lipids and sugars were compared. The lipid portion consisted of a homologous series of fatty acids ranging from C12:0 to C18:0 (predominant homologues, C12:0, C14:0 and C16:0) and the homologous series of 3-hydroxy acids ranging from C12:0 to C16:0 (predominant homologue, 3-OH-C14:0). The major sugar portion consisted of D-glucosamine. The toxicity of lipid A in mice (LD50) ranged between 300-400 micrograms/mouse, and values from the Limulus amebocyte lysate assay were recorded as titres of 10(-5) to 10(-6) mg/ml.

Characteristics of lipid A-protein complex from endotoxin of Shigella dysenteriae type 1 (S and R strains).

Mild acetic acid hydrolysis of endotoxin (lipopolysaccharide-protein complex) of Shigella dysenteriae type 1 (S and R forms) yielded a lipid A-protein complex that consisted of amino acids, fatty acids, and sugar and, in terms of chemical composition, displayed no marked differences between the S and R forms. Its protein portion (53 to 56%) consisted of at least 16 amino acids. In the fatty acid portion (14 to 18%), myristic, 3-hydroxymyristic, palmitic, and stearic acids accounted for 50%. The sugar portion (10 to 12%) consisted solely of glucosamine. The remainder was unidentified substances, most of which contained phosphorus. Lipid A-protein complexes derived from both S and R forms were not toxic for mice in doses up to 1,000 microgram/mouse, but their Linulus test activity had increased considerably as compared with the starting lipopolysaccharide-protein complex material: from 10(-6) to 10(-10--10(-12) mg/ml. The lipid A-protein complexes were readily soluble in a water solution of triethylamine, in dimethyl sulfoxide, and in pyridine.

[Synthesis of eumelanin pigment precursors. II. 6-hydroxy-5-methoxy- and 5-hydroxy-6- methoxyindole]. / Syntéza prekurzoru pigmentu eumelaninu. II. 6-hydroxy-5-methoxy- a 5-hydroxy-6-methoxyindol.

Following up of specific melanogenesis metabolite excretion in the course of malignant melanoma disease is useful for the disease prognosis assessment. The authors elaborated a modified synthesis technique of 6-hydroxy-5-methoxy and 5-hydroxy-6-methoxyindole, eumelanin pigment precursors. The elaborated synthesis is economically and temporally reasonable and the synthesized compounds accord, as proved by means of elementary analysis, thin layer (TLC) and high performance liquid chromatography (HPLC) and last but not least by nuclear magnetic resonance (NMR), with demands for reference compounds needed for isomer hydroxymethoxyindole quantification in urine.

[Synthesis of eumelanin pigment precursors. II. 6-Hydroxy-5-methoxy- and 5-hydroxy-6-methoxyindol]. / Syntéza prekurzoru pigmentu eumelaninu. II. 6-Hydroxy-5-methoxy-a 5-hydroxy-6-methoxyindol.

Following up of specific melanogenesis metabolite excretion in the course of malignant melanoma disease is useful for the disease prognosis assessment. The authors elaborated a modified synthesis technique of 6-hydroxy-5-methoxy and 5-hydroxy-6-methoxyindole, eumelanin pigment precursors. The elaborated synthesis is economically and temporally reasonable and the synthesized compounds accord, as proved by means of elementary analysis, thin layer (TLC) and high performance liquid chromatography (HPLC) and last but not least by nuclear magnetic resonance (NMR), with demands for reference compounds needed for isomer hydroxymethoxyindole quantification in urine.