Nanoscale Infrared Spectroscopic Characterization of Extended Defects in 4H-Silicon Carbide.

Extended defects in wide-bandgap semiconductors have been widely investigated using techniques providing either spectroscopic or microscopic information. Nano-Fourier transform infrared spectroscopy (nano-FTIR) is a nondestructive characterization method combining FTIR with nanoscale spatial resolution (∼20 nm) and topographic information. Here, we demonstrate the capability of nano-FTIR for the characterization of extended defects in semiconductors by investigating an in-grown stacking fault (IGSF) present in a 4H-SiC epitaxial layer. We observe a local spectral shift of the mid-infrared near-field response, consistent with the identification of the defect stacking order as 3C-SiC (cubic) from comparative simulations based on the finite dipole model (FDM). This 3C-SiC IGSF contrasts with the more typical 8H-SiC IGSFs reported previously and is exemplary in showing that nanoscale spectroscopy with nano-FTIR can provide new insights into the properties of extended defects, the understanding of which is crucial for mitigating deleterious effects of such defects in alternative semiconductor materials and devices.

A novel cricket morph has diverged in song and wing morphology across island populations.

Divergence of sexual signals between populations can lead to speciation, yet opportunities to study the immediate aftermath of novel signal evolution are rare. The recent emergence and spread of a new mating song, purring, in Hawaiian populations of the Pacific field cricket (Teleogryllus oceanicus) allows us to investigate population divergence soon after the origin of a new signal. Male crickets produce songs with specialized wing structures to attract mates from afar (calling) and entice them to mate when found (courtship). However, in Hawaii, these songs also attract an eavesdropping parasitoid fly (Ormia ochracea) that kills singing males. The novel purring song, produced with heavily modified wing morphology, attracts female crickets but not the parasitoid fly, acting as a solution to this conflict between natural and sexual selection. We've recently observed increasing numbers of purring males across Hawaii. In this integrative field study, we investigated the distribution of purring and the proportion of purring males relative to other morphs in six populations on four islands and compared a suite of phenotypic traits (wing morphology, calling song and courtship song) that make up this novel signal across populations of purring males. We show that purring is found in varying proportions across five, and is locally dominant in four, Hawaiian populations. We also show that calling songs, courtship songs and wing morphology of purring males differ geographically. Our findings demonstrate the rapid pace of evolution in island populations and provide insights into the emergence and divergence of new sexual signals over time.

Rectus Sheath Block Improves Patient Recovery Following Open Aortic Surgery.

BACKGROUND: Postoperative pain management remains a barrier to recovery following aortic surgery. Although epidural catheters help in adjunctive pain management, less is known about the use of rectus sheath blocks. We compared patient recovery following open abdominal aortic surgery (OAS) with and without adjunctive rectus block. METHODS: Adult patients undergoing open abdominal aortic aneurysm repair and aortobifemoral or aortoiliac bypass for occlusive disease were identified and stratified by use of general anesthesia alone (GA) versus adjunctive use of a rectus sheath block (GA + RB). A small number of patients with GA and concomitant epidural analgesia, along with patients that had retroperitoneal repairs, were not included in further analysis. Outcomes included time to extubation, intraoperative and postoperative morphine milligram equivalents (MME) utilization, length of stay, discharge MME, and postoperative complications. Categorial data were compared with Person Chi-Square tests or Fisher's exact tests. Continuous data were tested with independent t-tests or Mann-Whitney U-tests. RESULTS: From January 2017 to April 2022, there were 106 patients who underwent open aortic surgery, 55 patients with GA alone, 39 with GA + RB, and 12 patients who had a GA with concomitant epidural analgesia. Between GA and GA + RB, patients were comparable in both groups in terms of age, BMI (body mass index), smoking history, hypertension, diabetes, CAD (coronary artery disease), COPD (chronic obstructive pulmonary disease), and ASA (American Society of Anesthesiologists) class and prior opioid use. Patients with GA + RB were more likely to have scheduled elective procedures (80% GA cohort vs. 94.9% RB, P = 0.040), and a lower incidence of retroperitoneal exposure (14.5% GA cohort vs. 0% RB, P = 0.019). Patients with GA + RB had shorter time to extubation than GA (84.6% < 12 hr vs. 44.4%, P < 0.001), greater rate of procedural ketamine usage (GA + RB: 61.5% vs. GA: 40.0%, P = 0.049), lower MME at first postoperative day (median MME GA + RB: 25.0 vs. GA: 67.5, P = 0.002), lower discharge MME (median MME GA + RB: 142.5 vs. GA: 225.0, P = 0.036), and overall shorter length of stay (median stay GA + RB: 5 vs. GA: 6 days, P = 0.006). Postoperative complications were similar between groups. Similar findings were found in the comparison between elective-only GA and GA + RB patients and after exclusion of patients who only had a single shot of regional anesthesia. CONCLUSIONS: Patients that receive adjunctive rectus sheath blocks for pain control following OAS utilize fewer opioid medications during hospital stay and at discharge. Rectus sheath blocks represent an alternative option to other periprocedural analgesia following open aortic surgery.

Nitisinone Treatment Affects Biomarkers of Bone and Cartilage Remodelling in Alkaptonuria Patients.

Nitisinone has been approved for treatment of alkaptonuria (AKU). Non-invasive biomarkers of joint tissue remodelling could aid in understanding the molecular changes in AKU pathogenesis and how these can be affected by treatment. Serological and urinary biomarkers of type I collagen and II collagen in AKU were investigated in patients enrolled in the randomized SONIA 2 (NCT01916382) clinical study at baseline and yearly until the end of the study (Year 4). The trajectories of the biomarkers over time were observed. After treatment with nitisinone, the biomarkers of type I collagen remodelling increased at Year 1 (19% and 40% increase in CTX-I and PRO-C1, respectively), which was potentially reflected in the higher degree of mobility seen following treatment. The biomarkers of type II collagen remodelling decreased over time in the nitisinone group: C2M showed a 9.7% decline at Year 1, and levels then remained stable over the following visits; CTX-II showed a 26% decline at Year 3 and 4 in the nitisinone-treated patients. Nitisinone treatment induced changes in biomarkers of bone and cartilage remodelling. These biomarkers can aid patient management and deepen our knowledge of the molecular mechanisms of this rare disease.

Building a Healthcare Alliance for Resourceful Medicine Offensive Against Neoplasms in Hematology Added Value Framework for Hematologic Malignancies: A Comparative Analysis of Existing Tools.

OBJECTIVES: The Innovative Medicines Initiative-funded, multistakeholders project Healthcare Alliance for Resourceful Medicine Offensive Against Neoplasms in Hematology (HARMONY) created a task force involving patient organizations, medical associations, pharmaceutical companies, and health technology assessment/regulator agencies' representatives to evaluate the suitability of previously established value frameworks (VFs) for assessing the clinical and societal impact of new interventions for hematologic malignancies (HMs). METHODS: Since the HARMONY stakeholders identified the inclusion of patients' points of view on evaluating VFs as a priority, surveys were conducted with the patient organizations active in HMs and part of the HARMONY network, together with key opinion leaders, pharmaceutical companies, and regulators, to establish which outcomes were important for each HM. Next, to evaluate VFs against the sources of information taken into account (randomized clinical trials, registries, real-world data), structured questionnaires were created and filled by HARMONY health professionals to specify preferred data sources per malignancy. Finally, a framework evaluation module was built to analyze existing clinical VFs (American Society of Clinical Oncology, European Society of Medical Oncology, Magnitude of Clinical Benefit Scale, Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen, Institute for Clinical and Economic Review, National Comprehensive Cancer Network Evidence Blocks, and patient-perspective VF). RESULTS: The comparative analysis describes challenges and opportunities for the use of each framework in the context of HMs and drafts possible lines of action for creating or integrating a more specific, patient-focused clinical VF for HMs. CONCLUSIONS: None of the frameworks meets the HARMONY goals for a tool that applies to HMs and assesses in a transparent, reproducible, and systematic way the therapeutic value of innovative health technologies versus available alternatives, taking a patient-centered approach and using real-world evidence.

Female Sex is Associated with Worse Outcomes following Complex Fenestrated or Branched Endovascular Aortic Repair.

BACKGROUND: Sex-related discrepancies after standard endovascular aneurysm repair (EVAR) are noted to disproportionally affect females. A growing body of literature suggests similar disparities may extend to complex fenestrated or branched endovascular aneurysm repair (FBEVAR). However, recent examination of complex FBEVAR by a consortium of high-volume centers noted equivalent mortality among sexes. Whether similar results extend to non-trial data is unknown. METHODS: We examined all juxta-renal through type IV thoraco-abdominal aneurysms (sealing zones 6-8) which underwent elective FBEVAR within the Vascular Quality Initiative (VQI) database from January 2012 to December 2020. Urgent, symptomatic, ruptured, and staged cases were excluded, as were parallel stent grafts. Demographics, comorbid conditions, and technical factors were compared between sexes. Univariate analysis with Wilcoxon ranked sum tests and Chi-square tests of proportion were performed, followed by multivariate logistic regression for failure of target vessel technical success, reintervention, complications, and in-hospital mortality. RESULTS: Our analysis included 1,521 patients, 1,180 males (77.6%) and 341 females (22.4%). There were noted differences in pre-operative demographics, medical optimization, and technical aspects of the procedure. However, no difference was noted in proximal or distal sealing stents, number of fenestrations, or immediate endoleaks. On a multi variate logistic regression, female sex was an independent predictor of failure of target vessel technical success (odds ratio (OR) 3.339, 95% confidence interval (CI): 2.173-5.132, P < 0.001), reintervention (OR 2.192, 95% CI: 1.304-3.683, P = 0.003), complications (OR 1.747, 95% CI: 1.282-2.381, P < 0.001), and in-hospital mortality (OR 2.836, 95% CI: 1.510-5.328, P = 0.001). CONCLUSIONS: Females suffer worse outcomes after FBEVAR despite similar extent of disease, number of fenestrations, and incidence of immediate endoleak. Notable discrepancies were higher rates of chronic obstructive pulmonary disease (COPD) and lower rates of pre-operative aspirin, statin, and beta blocker therapy in females. Controlling for pre-operative demographics, female sex remained an independent predictor of worse outcomes. These discrepancies warrant further examination and should impact case planning for female patients undergoing complex aortic aneurysm repair.

Conditional targeting in mice reveals that hepatic homogentisate 1,2-dioxygenase activity is essential in reducing circulating homogentisic acid and for effective therapy in the genetic disease alkaptonuria.

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.

Intraoperative electroencephalographic changes during transcarotid artery revascularization are more frequent than previously reported.

OBJECTIVE: Up to 14% of patients undergoing carotid endarterectomy with continuous electroencephalographic (EEG) neuromonitoring will require shunt placement because of EEG changes. However, the initial studies of transcarotid artery revascularization (TCAR) found only one patient with temporary EEG changes. We report our experience with intraoperative EEG monitoring during TCAR. METHODS: We conducted a retrospective review of patients who underwent TCAR at two urban hospitals within an integrated healthcare network from May 2017 to January 2020. The data included demographic information, patient comorbidities, symptom status, previous carotid interventions, anatomic details, contralateral disease, intraoperative vital signs and EEG changes, and postoperative major adverse events (transient ischemic attack, stroke, myocardial infarction [MI], and death) both initially and at 30 days postoperatively. The Fisher exact test was used for categorical data and the Wilcoxon rank sum test for continuous data. RESULTS: A total of 89 patients underwent TCAR during the study period, of whom 71 (79.8%) received intraoperative EEG neuromonitoring. Of the 89 patients, 70.8% were men and 29.2% were women. The median age was 75 years (IQR, 68-82.5 years). Symptomatic patients accounted for 41.6% of the cohort. Of the 71 patients who received continuous neuromonitoring, 9 experienced EEG changes during TCAR (12.7%). The changes resolved in seven patients with pressure augmentation in three and switching to a low flow toggle in three. One patient who had sustained EEG changes had a new postoperative neurologic deficit. The median carotid stenosis percentage on preoperative computed tomography angiography was lower for patients with EEG changes than for those without (67% vs 80%; P = .01). No correlation was found between symptom status or 30-day stroke in patients with and without EEG changes (P = .49 and P = .24, respectively). Overall, three postoperative strokes, two postoperative deaths, and one MI occurred, for a composite 30-day stroke, death, and MI rate of 6.7%. CONCLUSIONS: Changes in continuous EEG monitoring were more frequent in our study than previously reported. Less severe carotid stenosis might be associated with a greater incidence of EEG changes. Limited data are available on the prognostic ability of EEG to detect clinically relevant changes during TCAR, and further studies are warranted.

"Lessons from Rare Forms of Osteoarthritis".

Osteoarthritis (OA) is one of the most prevalent conditions in the world, particularly in the developed world with a significant increase in cases and their predicted impact as we move through the twenty-first century and this will be exacerbated by the covid pandemic. The degeneration of cartilage and bone as part of this condition is becoming better understood but there are still significant challenges in painting a complete picture to recognise all aspects of the condition and what treatment(s) are most appropriate in individual causes. OA encompasses many different types and this causes some of the challenges in fully understanding the condition. There have been examples through history where much has been learnt about common disease(s) from the study of rare or extreme phenotypes, particularly where Mendelian disorders are involved. The often early onset of symptoms combined with the rapid and aggressive pathogenesis of these diseases and their predictable outcomes give an often-under-explored resource. It is these "rarer forms of disease" that William Harvey referred to that offer novel insights into more common conditions through their more extreme presentations. In the case of OA, GWAS analyses demonstrate the multiple genes that are implicated in OA in the general population. In some of these rarer forms, single defective genes are responsible. The extreme phenotypes seen in conditions such as Camptodactyly Arthropathy-Coxa Vara-pericarditis Syndrome, Chondrodysplasias and Alkaptonuria all present potential opportunities for greater understanding of disease pathogenesis, novel therapeutic interventions and diagnostic imaging. This review examines some of the rarer presenting forms of OA and linked conditions, some of the novel discoveries made whilst studying them, and findings on imaging and treatment strategies.

Anatomical Distribution of Ochronotic Pigment in Alkaptonuric Mice is Associated with Calcified Cartilage Chondrocytes at Osteochondral Interfaces.

Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl's staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c Hgd-/-, Hgd tm1a-/-), using Schmorl's staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (p = 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.

Characterising the arthroplasty in spondyloarthropathy in a large cohort of eighty-seven patients with alkaptonuria.

Arthroplasty in the spondyloarthropathy (SPOND) of alkaptonuria (AKU) in incompletely characterised. The aim was to improve the understanding of arthroplasty in AKU through a study of patients attending the National Alkaptonuria Centre (NAC). Eighty-seven patients attended the NAC between 2007 and 2020. Seven only attended once. Fifty-seven attended more than once and received nitisinone 2 mg daily. Twenty-three attended at least twice without receiving nitisinone. Assessments including questionnaire analysis eliciting details of arthroplasty and other surgical treatments for SPOND, 18 FPETCT and CT densitometry at the neck of hip and lumbar spine, as well as photographs of the eyes and ears were acquired from patients attending the National Alkaptonuria Centre (NAC) at baseline when 2 mg nitisinone was commenced, and yearly thereafter. Photographs were scored to derive ochronosis scores. Blood and urine samples were collected for chemical analyses. The prevalence of arthroplasty was 36.8%, similar in males and females, occurring especially in the knees, hips and shoulders. Multiple arthroplasties were found in 29 patients (33.3%) in this cohort. Incident arthroplasty was 6.5% in the nitisinone group and 7.1% in the no-nitisinone group. Incident arthroplasty was 11.3% in the group with baseline arthroplasty and 3.51% in the group without. A strong association of arthroplasty with SPOND (R = 0.5; P << .0001) and ochronosis (R = 0.54; P < .0001) was seen. Nitisinone had no significant effect on incident arthroplasty. Arthroplasty due to ochronosis and SPOND is common in AKU. Nitisinone decreased ochronosis but had no effect on arthroplasty in this cohort.

Characterizing the alkaptonuria joint and spine phenotype and assessing the effect of homogentisic acid lowering therapy in a large cohort of 87 patients.

A large alkaptonuria (AKU) cohort was studied to better characterize the poorly understood spondyloarthropathy of rare disease AKU. Eighty-seven patients attended the National Alkaptonuria Centre (NAC) between 2007 and 2020. Seven only attended once. Fifty-seven attended more than once and received nitisinone 2 mg daily. Twenty-three attended at least twice without receiving nitisinone. Assessments included questionnaire analysis, 18F Positron emission tomography computerised tomography (PETCT), as well as photographs of ochronotic pigment in eyes and ears at baseline when 2 mg nitisinone was commenced and yearly thereafter. Blood and urine samples were collected for chemical measurement. The prevalence of ochronosis, as well as pain, PETCT and combined pain and PETCT scores, was greatly increased at 90.5%, 85.7%, 100%, and 100%, respectively. Joint pain scores were greatest in proximal joints in upper and lower limbs. PETCT joint scores were higher in proximal joints in upper limb but higher in distal joints in the lower limb. Spine pain scores were highest in lumbar, followed by cervical, thoracic, and cervical regions at 77.4%, 59.5%, 46.4%, and 25%, respectively. PETCT spine scores were highest in thoracic followed by lumbar, cervical, and sacroiliac regions at 74.4%, 70.7%, 64.6%, and 47.8% respectively; ochronosis associated closely with spondyloarthropathy scores (R = .65; P < .0001). Nitisinone reversed ochronosis significantly, with a similar pattern of decreased joint and spine disease. Spondyloarthropathy is a highly prevalent feature in this NAC cohort. Ochronosis appears to be associated with spondyloarthropathy. Nitisinone decreases ochronosis and had a similar nonsignificant effect pattern on spondyloarthropathy.

An unusual example of hereditary multiple exostoses: a case report and review of the literature.

BACKGROUND: Hereditary multiple exostoses (HME) is a rare skeletal disorder characterised by a widespread. distribution of osteochondromas originating from the metaphyses of long bones. CASE PRESENTATION: This case study examines a 55-year-old male cadaver bequeathed to the University of Liverpool who suffered from HME, thus providing an exceptionally rare opportunity to examine the anatomical changes associated with this condition. CONCLUSIONS: Findings from imaging and dissection indicated that this was a severe case of HME in terms of the quantity and distribution of the osteochondromas and the number of synostoses present. In addition, the existence of enchondromas and the appearance of gaps within the trabeculae of affected bones make this a remarkable case. This study provides a comprehensive overview of the morbidity of the disease as well as adding to the growing evidence that diseases concerning benign cartilaginous tumours may be part of a spectrum rather than distinct entities.

Dietary restriction of tyrosine and phenylalanine lowers tyrosinemia associated with nitisinone therapy of alkaptonuria.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700 µmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. Elevated tyrosine (813 µmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3, 274.8, and 144.3 µmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3, 530.2, and 656.2 µmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (P = .002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine <700 µmol/L. Tyrosine/phenylalanine dietary restriction significantly reduced nitisinone-induced tyrosinemia in mice, with phenylalanine restriction alone proving ineffective. Similarly, protein restriction significantly reduced circulating tyrosine in AKU patients.

Nitisinone causes acquired tyrosinosis in alkaptonuria.

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[13 C9 ]tyrosine and l-[d8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P < .001), suggesting that NTBC increases tyrosine not just in serum but also in tissues (ie, acquired tyrosinosis). This study provides, for the first time, the experimental proof for the magnitude of NTBC-related acquired tyrosinosis which should be overcome to ensure the safe use of NTBC in AKU.

Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration.

Pigmentation Chemistry and Radical-Based Collagen Degradation in Alkaptonuria and Osteoarthritic Cartilage.

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA-derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone-resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation.

A Comprehensive LC-QTOF-MS Metabolic Phenotyping Strategy: Application to Alkaptonuria.

BACKGROUND: Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia. METHODS: A total of 619 standards (molecular weight, 45-1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods-2 reversed phase and 1 normal phase-coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min. RESULTS: Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD -/- mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration. CONCLUSIONS: The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU.

Ochronotic pigmentation is caused by homogentisic acid and is the key event in alkaptonuria leading to the destructive consequences of the disease-A review.

Ochronosis is the process in alkaptonuria (AKU) that causes all the debilitating morbidity. The process involves selective deposition of homogentisic acid (HGA)-derived pigment in tissues altering the properties of these tissues, leading to their failure. Some tissues like cartilage are more easily affected by ochronosis while others such as the liver and brain are unaffected for reasons that are still not understood. In vitro and mouse models of ochronosis have confirmed the dose relationships between HGA and ochronosis and also their modulation by p-hydroxyphenylpyruvate dioxygenase inhibition. Ochronosis cannot be fully reversed and is a key factor in influencing treatment decisions. Earlier detection of ochronosis preferably by noninvasive means is desirable. A cause-effect relationship between HGA and ochronosis is discussed. The similarity in AKU and familial hypercholesterolaemia is explored and lessons learnt. More research is needed to more fully understand the crucial nature of ochronosis.

Alkaptonuria: An example of a "fundamental disease"--A rare disease with important lessons for more common disorders.

"Fundamental diseases" is a term introduced by the charity Findacure to describe rare genetic disorders that are gateways to understanding common conditions and human physiology. The concept that rare diseases have important lessons for biomedical science has been recognised by some of the great figures in the history of medical research, including Harvey, Bateson and Garrod. Here we describe some of the recently discovered lessons from the study of the iconic genetic disease alkaptonuria (AKU), which have shed new light on understanding the pathogenesis of osteoarthritis. In AKU, ochronotic pigment is deposited in cartilage when collagen fibrils become susceptible to attack by homogentisic acid (HGA). When HGA binds to collagen, cartilage matrix becomes stiffened, resulting in the aberrant transmission of loading to underlying subchondral bone. Aberrant loading leads to the formation of pathophysiological structures including trabecular excrescences and high density mineralised protrusions (HDMPs). These structures initially identified in AKU have subsequently been found in more common osteoarthritis and appear to play a role in joint destruction in both diseases.