Quantification of the flux of tyrosine pathway metabolites during nitisinone treatment of Alkaptonuria.

Nitisinone decreases homogentisic acid (HGA) in Alkaptonuria (AKU) by inhibiting the tyrosine metabolic pathway in humans. The effect of different daily doses of nitisinone on circulating and 24 h urinary excretion of phenylalanine (PA), tyrosine (TYR), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and HGA in patients with AKU was studied over a four week period. Forty AKU patients, randomised into five groups of eight patients, received doses of 1, 2, 4 or 8 mg of nitisinone daily, or no drug (control). Metabolites were analysed by tandem mass spectrometry in 24 h urine and serum samples collected before and after nitisinone. Serum metabolites were corrected for total body water and the sum of 24 hr urine plus total body water metabolites of PA, TYR, HPPA, HPLA and HGA were determined. Body weight and urine urea were used to check on stability of diet and metabolism over the 4 weeks of study. The sum of quantities of urine metabolites (PA, TYR, HPPA, HPLA and HGA) were similar pre- and post-nitisinone. The sum of total body water metabolites were significantly higher post-nitisinone (p < 0.0001) at all doses. Similarly, combined 24 hr urine:total body water ratios for all analytes were significantly higher post-nitisinone, compared with pre-nitisinone baseline for all doses (p = 0.0002 - p < 0.0001). Significantly higher concentrations of metabolites from the tyrosine metabolic pathway were observed in a dose dependant manner following treatment with nitisinone and we speculate that, for the first time, experimental evidence of the metabolite pool that would otherwise be directed towards pigment formation, has been unmasked.

Data on items of AKUSSI in Alkaptonuria collected over three years from the United Kingdom National Alkaptonuria Centre and the impact of nitisinone.

Alkaptonuria is a rare genetic disorder characterized by a high level of circulating (and urine) homogentisic acid (HGA), which contributes to ochronosis when it is deposited in connective tissue as a pigmented polymer. In an observational study carried out by National AKU Centre (NAC) in Liverpool, a total of thirty-nine AKU patients attended yearly visits in varying numbers. At each visit a mixture of clinical, joint and spinal assessments were carried out and the results calculated to yield an AKUSSI (Alkaptonuria Severity Score Index), see "Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre" (Ranganath at el., 2018). The aim of this data article is to produce visual representation of the change in the components of AKUSSI over 3 years, through radar charts. The metabolic effect of nitisinone is shown through box plots.

Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: Evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre.

QUESTION: Does Nitisinone prevent the clinical progression of the Alkaptonuria? FINDINGS: In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period. MEANING: Nitisinone is a beneficial therapy in Alkaptonuria. BACKGROUND: Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU). METHODS: Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at V1 or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre-baseline visit (V0) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended V0, V1, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases. RESULTS: The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/month, in the SAME group, was significantly lower at two (0.32 ±â€¯0.19) and three (0.15 ±â€¯0.13) years post-nitisinone when compared to pre-nitisinone (0.65 ±â€¯0.15) (p < .01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 ±â€¯0.08) and three (0.19 ±â€¯0.06) years post-nitisinone when compared to pre-nitisinone (0.59 ±â€¯0.13) (p < .01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three year's post-nitisinone in both VAR and SAME groups compared with pre-nitisinone (p < .05). CONCLUSION: This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone.

Expression of dihydropyrimidine dehydrogenase (DPD) and hENT1 predicts survival in pancreatic cancer.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) tumour expression may provide added value to human equilibrative nucleoside transporter-1 (hENT1) tumour expression in predicting survival following pyrimidine-based adjuvant chemotherapy. METHODS: DPD and hENT1 immunohistochemistry and scoring was completed on tumour cores from 238 patients with pancreatic cancer in the ESPAC-3(v2) trial, randomised to either postoperative gemcitabine or 5-fluorouracil/folinic acid (5FU/FA). RESULTS: DPD tumour expression was associated with reduced overall survival (hazard ratio, HR = 1.73 [95% confidence interval, CI = 1.21-2.49], p = 0.003). This was significant in the 5FU/FA arm (HR = 2.07 [95% CI = 1.22-3.53], p = 0.007), but not in the gemcitabine arm (HR = 1.47 [0.91-3.37], p = 0.119). High hENT1 tumour expression was associated with increased survival in gemcitabine treated (HR = 0.56 [0.38-0.82], p = 0.003) but not in 5FU/FA treated patients (HR = 1.19 [0.80-1.78], p = 0.390). In patients with low hENT1 tumour expression, high DPD tumour expression was associated with a worse median [95% CI] survival in the 5FU/FA arm (9.7 [5.3-30.4] vs 29.2 [19.5-41.9] months, p = 0.002) but not in the gemcitabine arm (14.0 [9.1-15.7] vs. 18.0 [7.6-15.3] months, p = 1.000). The interaction of treatment arm and DPD expression was not significant (p = 0.303), but the interaction of treatment arm and hENT1 expression was (p = 0.009). CONCLUSION: DPD tumour expression was a negative prognostic biomarker. Together with tumour expression of hENT1, DPD tumour expression defined patient subgroups that might benefit from either postoperative 5FU/FA or gemcitabine.

Intratumoural expression of deoxycytidylate deaminase or ribonuceotide reductase subunit M1 expression are not related to survival in patients with resected pancreatic cancer given adjuvant chemotherapy.

BACKGROUND: Deoxycytidylate deaminase (DCTD) and ribonucleotide reductase subunit M1 (RRM1) are potential prognostic and predictive biomarkers for pyrimidine-based chemotherapy in pancreatic adenocarcinoma. METHODS: Immunohistochemical staining of DCTD and RRM1 was performed on tissue microarrays representing tumour samples from 303 patients in European Study Group for Pancreatic Cancer (ESPAC)-randomised adjuvant trials following pancreatic resection, 272 of whom had received gemcitabine or 5-fluorouracil with folinic acid in ESPAC-3(v2), and 31 patients from the combined ESPAC-3(v1) and ESPAC-1 post-operative pure observational groups. RESULTS: Neither log-rank testing on dichotomised strata or Cox proportional hazard regression showed any relationship of DCTD or RRM1 expression levels to survival overall or by treatment group. CONCLUSIONS: Expression of either DCTD or RRM1 was not prognostic or predictive in patients with pancreatic adenocarcinoma who had had post-operative chemotherapy with either gemcitabine or 5-fluorouracil with folinic acid.

Evaluation in pre-diagnosis samples discounts ICAM-1 and TIMP-1 as biomarkers for earlier diagnosis of pancreatic cancer.

Circulating intercellular adhesion molecule-1 (ICAM-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) have been widely proposed as potential diagnostic biomarkers for pancreatic ductal adenocarcinoma (PDAC). We report on serum protein levels prior to clinical presentation of pancreatic cancer. Serum ICAM-1 and TIMP-1 were measured by ELISA in two case­control sets: 1) samples from patients diagnosed with pancreatic cancer (n = 40), chronic pancreatitis (n = 20), benign jaundice due to gall stones (n = 20) and healthy subjects (n = 20); 2) a preclinical set from the UK Collaborative Trial of Ovarian Cancer Screening biobank of samples collected from 27 post-menopausal women 0­12 months prior to diagnosis of pancreatic cancer and controls matched for date of donation and centre. Levels of ICAM-1 and TIMP-1 were significantly elevated in set 1 in PDAC patients with jaundice compared to PDAC patients without jaundice and both proteins were elevated in patients with jaundice due to gall stones. Neither protein was elevated in samples taken 0­12 months prior to PDAC diagnosis compared to non-cancer control samples. In conclusion, evaluation in pre-diagnosis samples discounts ICAM-1 and TIMP-1 as biomarkers for earlier diagnosis of pancreatic cancer. Failure to account for obstructive jaundice may have contributed to the previous promise of these candidate biomarkers. BIOLOGICAL SIGNIFICANCE: Pancreatic cancer is usually diagnosed when at an advanced stage which greatly limits therapeutic options. Biomarkers that could facilitate earlier diagnosis are urgently sought.

Does the 'Liverpool Care Pathway' facilitate an improvement in quality of care for dying cancer patients?

BACKGROUND: The Liverpool Care Pathway for the Dying Patient (LCP) aims to transfer hospice principles of care for dying patients to other health-care sectors. This post-bereavement survey explored the LCP's effectiveness in improving quality of care for cancer patients. METHODS: Postal self-completion questionnaires were sent to 778 next-of-kin to consecutive deceased patients who had died an 'expected' cancer death in a hospice and acute tertiary hospital. RESULTS: Following exclusions (n=53), 255 of the 725 next-of-kin agreed to participate (35.2% response rate). Overall hospice participants reported the best quality of care, and hospital participants, for whom care was not supported by the LCP, reported the worst quality of care. Multivariate analysis showed the hospice was an independent predictor for patients being treated with dignity (OR 8.46) and receiving adequate family support (OR 7.18) (P<0.0001). Care supported by the LCP and the hospital specialist palliative care team were both associated with good family support, but neither was an independent predictor. CONCLUSIONS: From the bereaved relatives' perspective, within the hospital, the LCP is effective in improving specific aspects of care, such as symptom control for dying patients. Further improvement is required, however, to attain the hospice standard of care.

The measurement of enamel wear by four toothpastes.

OBJECTIVES: To determine the enamel abrasivity of three whitening toothpastes and a silica toothpaste and to measure the brushing forces used. METHODS: Polished human enamel blocks were indented with a Knoop diamond and attached to dentures of adult volunteers. The blocks were brushed ex vivo, twice per day with either a whitening toothpaste containing Perlite (White System), a commercial whitening toothpaste (A and B) or a silica toothpaste. After four and twelve-weeks, one block per subject was removed and the Knoop indent remeasured. From the changes in the indent length, the amount of enamel wear was calculated. RESULTS: The mean enamel wear (sd) for White System, silica toothpaste, whitening toothpaste A and B after four-weeks was 0.14 (0.15), 0.09 (0.16), 0.14 (0.12) and 0.89 (0.93) and after twelve-weeks was 0.24 (0.21), 0.37 (0.73), 0.36 (0.52) and 1.04 (0.98) microm respectively. After four-weeks, the differences in enamel wear between whitening toothpaste B and all other toothpastes were of statistical significance (p < 0.003), but not after twelve-weeks. The mean ex vivo normal brushing force (sd) was 2.67 (1.33) N. CONCLUSIONS: All three whitening toothpastes did not give significantly more enamel wear than a silica toothpaste after twelve-weeks in situ with ex vivo brushing.

Abrasion of human enamel and dentine caused by toothpastes of differing abrasivity determined using an in situ wear model.

OBJECTIVES: To determine the rates of enamel and dentine wear in situ during tooth brushing with a range of five toothpastes of differing RDA and REA values. METHODS: Enamel/dentine inserts were placed in the buccal aspects of full upper dentures of human volunteers. Enamel wear was determined by change in Knoop indent length and dentine wear was determined from the enamel-dentine step height, measured using optical profilometry. RESULTS: Significant differences between the test products were found for both enamel and dentine (p < 0.05). However for the conventional toothpastes the rate of wear was similar and differences between the products were not generally statistically significant for either enamel or dentine, despite the fact that their RDA values differed by more than a factor of two. The rate of wear was found to decrease throughout the duration of the study. CONCLUSIONS: The study demonstrated that the methodology used is capable of assessing enamel and dentine wear in situ within a relatively short period of time. The data suggest that whilst in vitro tests have value in understanding differences in abrasivity between products, they cannot be used to predict in vivo effects.

The measurement of enamel and dentine abrasion by tooth whitening products using an in situ model.

OBJECTIVES: To determine the enamel and dentine wear of two whitening toothpastes using an in situ model with ex vivo brushing. METHODS: Human enamel/dentine (approximately 50:50) blocks (approximately 4 x 4mm) were placed in the upper buccal aspects of full or partial dentures of a group of 25 subjects. Subjects brushed the specimens ex vivo with either a calcium carbonate/perlite or silica containing whitening toothpaste under exaggerated conditions as compared to normal for 30 s, twice per day. Specimens were removed after 4, 8 and 12 weeks and the wear to the enamel and dentine was determined. Enamel wear was determined by change in Knoop indent length and dentine wear was determined from the enamel-dentine step height, measured using optical profilometry. RESULTS: The mean wear after 12 weeks was for enamel 0.27 and 0.19 microns, and for dentine 34.3 and 61.1 microns, for the calcium carbonate/perlite and silica toothpastes respectively. There were no significant differences between products after 12 weeks. The rate of wear was found to decrease throughout the duration of the study. CONCLUSION: There were no significant differences between the two whitening toothpastes in terms of enamel and dentine wear after 12 weeks brushing.

Effect of toothpaste with natural calcium carbonate/perlite on extrinsic tooth stain.

OBJECTIVE: The current study was designed to determine the effect of natural calcium carbonate toothpaste containing Perlite and microgranules (Whitening toothpaste) on extrinsic tooth stain compared to a standard commercial toothpaste formulation with precipitated calcium carbonate (PCC) as abrasive and a commercial toothpaste with dicalcium phosphate dihydrate (DCPD) as abrasive. METHODS: The toothpastes were evaluated in a double blind, three-cell, stratified (tobacco use; baseline tooth stain level), parallel group design study involving 600 subjects with extrinsic tooth stain. Subjects brushed twice daily with their allocated toothpaste for four weeks. Extrinsic tooth stain was measured using the Macpherson modification of the Lobene stain index. RESULTS: ANCOVA showed significant differences between toothpastes (p=0.037). Subsequent multiple comparisons using pairwise t-tests, showed the Whitening toothpaste to be superior to the DCPD toothpaste (p=0.014) and the PCC toothpaste (p=0.067). When a Box-Cox transformation was made to the data (y0.6) to improve normality, these two differences were more accurately estimated at p=0.004 and p=0.03 respectively. CONCLUSION: The Whitening toothpaste has been shown to be significantly more effective in tooth stain removal than the two standard commercial toothpaste formulations.

The effect of a 2% zinc citrate, 0.3% Triclosan dentifrice on plaque acid production following consumption of a snackfood.

OBJECTIVES: A) To assess plaque lactate production following consumption of three foods (cake, chocolate/caramel bar, sweetened coffee), and B) To measure the effect of a fluoride dentifrice containing 2% zinc citrate and 0.3% Triclosan on plaque lactate and pH drop following consumption of cake. METHODS: A) 10 subjects completed the first study. Plaque samples taken before and at 8,15 and 30 minutes after eating. Samples were analysed for lactate via Capillary Electrophoresis. B) 30 subjects completed the second study. Plaque samples were taken before and after cake and use of test dentifrice or no treatment control. Plaque pH and lactate content were assessed. RESULTS: A) Plaque lactate levels increased after all three foods; peak lactate levels occurred 8 minutes after eating. B) Plaque lactate concentrations after eating cake were 39.2mM for the control treatment and a significantly lower value, 23.6mM, for the test 2% zinc citrate, 0.3% Triclosan dentifrice. After food challenge, pH values were 5.53 for the no treatment group and a significantly higher value of 5.79 for the test dentifrice group. CONCLUSIONS: A toothpaste containing 2% zinc citrate, 0.3% Triclosan can significantly reduce plaque lactate generation and pH drop induced by cake, compared to no treatment control.

The effect of a toothpaste containing 2% zinc citrate and 0.3% Triclosan on bacterial viability and plaque growth in vivo compared to a toothpaste containing 0.3% Triclosan and 2% copolymer.

OBJECTIVE: To compare the antimicrobial efficacy and effect on plaque growth of a new silica-based fluoride toothpaste containing 2% zinc citrate/ 0.3% Triclosan with a silica-based fluoride toothpaste containing 0.3% Triclosan/2% copolymer. METHODS: In Study 1, plaque was collected after one week's use of each toothpaste and assessed for bacterial viability, live/ dead ratio and microbial membrane integrity. In study 2, plaque was measured immediately and 18 hours after a single brushing with the specified toothpastes. RESULTS: The 2% zinc citrate/0.3% Triclosan formulation significantly reduced the total number of viable aerobic and anaerobic bacteria (p = 0.0223 and p = 0.0443 respectively) compared to the 0.3% Triclosan/2% copolymer formulation. Both toothpastes increased the bacterial membrane permeability significantly. However, the proportion of live bacteria for the 2% zinc citrate/0.3% Triclosan product was significantly reduced (p < 0.05). Study 2 showed significantly less plaque growth 18 hours after using the 2% zinc citrate/0.3% Triclosan toothpaste compared to the 0.3% Triclosan/2% copolymer toothpaste (p < 0.01). CONCLUSION: Regular use of a fluoride toothpaste containing 2% zinc citrate and 0.3% Triclosan, significantly reduced the viability of plaque bacteria compared to a fluoride toothpaste containing 0.3% Triclosan/ 2% copolymer 12 hours after brushing. In addition, a clinical plaque growth study confirmed that this anti-microbial efficacy leads to a significant reduction in plaque growth.

Prospective study of the hypothalamic-pituitary axis in thalassaemic patients who developed secondary amenorrhoea.

OBJECTIVE: We aimed to prospectively evaluate during 10 years the GnRH-gonadotrophin secretory dynamics in a cohort of 15 menstruating girls with beta-thalassaemia major to determine whether they sustained progressive damage to this axis. DESIGN: Patients were characterized by 12-hour gonadotrophin profiles (by sampling blood at 15-minute intervals) and assessment of gonadotrophin responses to 100 micrograms GnRH bolus (by sampling blood at 20-minute intervals for 1 hour and at 2 hours) sequentially during the follicular and luteal phases of their menstrual cycles, 12-14 months and 5-6 years after the onset of secondary amenorrhoea. SUBJECTS: We studied 15 post-menarcheal thalassaemic girls and five age-matched control subjects who were the healthy siblings of the patients. MEASUREMENTS: FSH and LH assays were determined using commercial RIA systems and double antibody techniques. Pulse detection used the Pulsar technique of Merriam and Wachter. RESULTS: We demonstrated that during their amenorrhoeic period, all thalassaemic patients had gonadotrophin pulse abnormalities and low-normal GnRH-stimulated gonadotrophin levels indicating that they had GnRH-gonadotrophin secretory insufficiency. During the subsequent 10 years there was progressive deterioration of hypothalamic-pituitary function in all patients; 66% became apulsatile and all had marked reduction in their GnRH-stimulated gonadotrophin levels. CONCLUSIONS: Our investigation suggests that thalassaemic patients with secondary amenorrhoea had severe and progressive damage to their hypothalamic-pituitary axes despite intensive chelation therapy.

Time-series analysis of urethral electrical conductance measurements in the assessment of unstable urethral pressure: results in normal patients and in those with genuine stress incontinence.

It is well established that urethral pressure variations occur in patients with or without urinary incontinence, but to what extent they contribute to a patient's symptoms remains unclear. Previous work has suggested that in stress incontinent patients, a rise in bladder neck electrical conductance (BNEC) occurs simultaneously with a fall in urethral pressure, and that this represents bladder neck opening. Six patients with genuine stress incontinence (gsi), and six normal controls underwent simultaneous urethral pressure and BNEC measurements, the results being subjected to time-series analysis, to determine whether the previous finding could be confirmed statistically, and to establish whether a similar association was present in normal women. All six patients with gsi and five of the normal patients had unstable urethral pressure. Estimated cross-correlation of differenced data for the two parameters showed a significant negative correlation at zero time lag in three patients with gsi and two of the control group. There was no significant correlation between the two parameters in the other seven patients. We conclude that the previous finding of a correlation between urethral pressure variation and BNEC, suggesting bladder neck opening occurs as urethral pressure falls in patients with gsi is confirmed, but appeared to be present in only 50% of patients; the finding is just as likely in normal patients, and therefore whilst it may be of relevance to the severity of symptoms, could not be held to have any aetiological significance.

Computer modelling of ENT out-patients.

The behaviour of queues in ENT out-patients has been simulated by a computer program. This allowed various appointment systems to be studied and improved to allow a smooth flow of patients through the clinic, in such a way that the idle time of the clinic staff is reduced without increasing patient waiting times.