Anna Milan

Vitamin D Assays: Past and Present Debates, Difficulties, and Developments

Clinical interest in Vitamin D and its purported roles not only in calcium and bone metabolism but in several other medical conditions (diabetes, cardiovascular disease, multiple sclerosis, cancer, psychiatric disorders, neuro-muscular disease) has led to a surge in laboratory requests for 25 hydroxy vitamin D and 1,25 dihydroxy vitamin D measurement. Circulating 25 hydroxy vitamin D concentration is routinely used as the best indicator of vitamin D status, but measurement of other metabolites, especially the physiologically active 1,25 dihyroxy vitamin D, are of clinical value. Over the last 40 years the development of assays for vitamin D and its metabolites from early competitive binding assays through to immunoassay and liquid chromatography aligned to mass spectrometry have demonstrated various analytical challenges, the advantages and disadvantages of each method are constantly changing with new technological developments. Immunoassay remains the predominant mode of measurement for 25-hydroxy vitamin D although problems with equimolar recovery of the D2 and D3 metabolites remain an issue. Standardisation of all assays has been improved but not resolved with the currently available reference materials as evidenced by the international vitamin D external quality assurance scheme, DEQAS. The choice of method for each laboratory remains a balance mainly between turn around time, convenience, cost and the specificity and accuracy of the information obtained. With increasing discussion and clinical interest surrounding other vitamin D metabolites the vitamin D assay debate is set to continue.

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment

Background Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. Methods Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. Findings A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. Conclusions In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. Trial registration number EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

Molecular interaction of recombinant decorin and biglycan with type I collagen influences crystal growth

This study has investigated the interaction of recombinant chondroitin sulphate (CS)-substituted decorin and biglycan on collagen fibrillogenesis, their interaction with hydroxyapatite (HAP), and HAP-induced crystal growth. The core proteins of the recombinant decorin and biglycan were obtained following chondroitinase ABC digestion and their influence on the above physical mechanisms were investigated in parallel. CS-decorin promoted collagen fibrillogenesis, with the interaction mediated principally through the core protein. Both decorin and biglycan demonstrated a strong association for HAP, predominately facilitated through the glycosaminoglycan chains. HAP-induced crystal growth was inhibited by decorin and biglycan, although the degree of inhibition was reduced when these proteoglycans were complexed with type I collagen. Thus, this study has highlighted potentially differing roles for decorin and biglycan, as both promoters and inhibitors in the regulation of the mineralization process.

Modulation of Collagen Fibrillogenesis by Dentinal Proteoglycans

Studies have identified different pools of proteoglycan (PG) species present within the unmineralized matrix of the predentine, the transitional phase at the predentine–dentine interface and the mineralized dentine. These PGs alter with respect to the chemical nature of their glycosaminoglycan (GAG) chains and as a result of extracellular processing of the macromolecule in the matrix. The present study has examined the influence of the PGs isolated from these phases and the influence of the attached GAG chains, upon their ability to control collagen fibrillogenesis. PGs isolated from the three phases were characterized and determined to contain a mixture of decorin and biglycan. Results have indicated that predentine PGs, which are substituted with a higher proportion of dermatan sulfate, significantly delayed fibril formation while ultimately promoting the formation of thicker fibrils. Removal of the GAG chains further delayed fibrillogenesis, leading to the formation of thinner fibrils, compared with the collagen-only control. PGs isolated from predentine–dentine, which contained a higher proportion of chondroitin sulfate, also significantly delayed fibrillogenesis, resulting in thicker collagen fibrils. GAG chains attached to the predentine–dentine interface PGs played a role in the timing of fibrillogenesis with fibril formation initiated at the same time as the collagen control, but yielding thicker fibrils. Dentine PGs significantly inhibited fibrillogenesis and fibril thickness over concentrations of 50–25 μg/mL protein. In conclusion, the PGs isolated from the distinct phases have indicated differing roles in the orchestrated organization of the extracellular matrix during dentinogenesis, with roles for both the core protein and attached GAG chains indicated.

Altered phosphorylation of rat dentine phosphoproteins by fluoride in vivo.

Abstract. Dentine phosphoproteins have been proposed to have an important role in mineralization. This study focused on the influence of fluoride on the biochemical composition of dentine phosphoproteins and attempts to relate changes to the altered mineralization witnessed during fluorosis. Wistar rats were rendered fluorotic by the administration of 20 ppm sodium fluoride in their drinking water ad libitum, a nonfluorotic group received double-distilled, deionized water only. After 17 weeks, the teeth showed signs of fluorosis. The incisors were removed, split longitudinally, and the pulps were removed. Teeth were powdered and demineralized in 10% EDTA with protease inhibitors, after which the organic matrix was extracted with 4 M guanidinium chloride. Phosphoproteins were selectively precipitated from the soluble extract by the addition of 1.0 M calcium chloride and further purified by anion exchange chromatography. SDS-PAGE revealed two protein bands with molecular weights of 130 kDa and 66 kDa in the nonfluorotic fraction and 116 kDa and 66 kDa in the fluorotic fraction. Western blotting analysis identified the 66 kDa band as α2-HS glycoprotein which co-precipitated with phosphoproteins. Electroelution of the protein bands was performed with subsequent biochemical analyses. Phosphate content was determined for each protein band and was detectable in the 116 kDa and 130 kDa bands from the fluorotic and nonfluorotic samples, respectively, with a decreased level noted in the 116 kDa band. The presence of phosphate and the amino acid analysis of these bands suggested their identity to be dentine phosphoproteins. No changes in the ratio of amino acids was detected in fluorotic samples. The fluoride-induced alterations to the biochemical structure of dentine phosphoproteins would appear to influence the phosphorylation of these macromolecules only, possibly affecting posttranslational events. Such alterations may play a role in disrupting the patterns of mineralization seen during fluorosis.

Analysis of HGD Gene Mutations in Patients with Alkaptonuria from the United Kingdom: Identification of Novel Mutations

Alkaptonuria (AKU) is a rare autosomal recessive disorder with incidence ranging from 1:100,000 to 1:250,000. The disorder is caused by a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGD), which results from defects in the HGD gene. This enzyme converts homogentisic acid to maleylacetoacetate and has a major role in the catabolism of phenylalanine and tyrosine. To elucidate the mutation spectrum of the HGD gene in patients with alkaptonuria from 42 patients attending the National Alkaptonuria Centre, 14 exons of the HGD gene and the intron-exon boundaries were analysed by PCR-based sequencing. A total of 34 sequence variants was observed, confirming the genetic heterogeneity of AKU. Of these mutations, 26 were missense substitutions and four splice site mutations. There were two deletions and one duplication giving rise to frame shifts and one substitution abolishing the translation termination codon (no stop). Nine of the mutations were previously unreported novel variants. Using computational approaches based on the 3D structure, these novel mutations are predicted to affect the activity of the protein complex through destabilisation of the individual protomer structure or through disruption of protomer-protomer interactions.

Fluoride alters casein kinase II and alkaline phosphatase activity in vitro with potential implications for dentine mineralization

Dentine phosphoprotein (DPP), a major non-collagenous acidic protein of dentine, undergoes altered phosphorylation in vivo in the presence of high fluoride concentrations. This has major implications for the altered mineralization patterns found during fluorosis. In dentine, casein kinase II is involved in phosphorylating DPP, and alkaline phosphatase (ALP) is ascribed roles in the dephosphorylation of DPP, increasing the inorganic phosphate at the mineralization front and the removal of pyrophosphate. Here the influence of fluoride in vitro on the activity of purified casein kinase II and ALP and its relation to altered patterns of mineralization were examined. Kinetic analysis showed that casein kinase II activity was completely inhibited at 0.04 M NaF. Vmax when compared to the control assay was significantly decreased (P < 0.0001) between concentrations 4 x 10(-4)-4 x 10(-8) M NaF. Significant changes to the Km (P < 0.0001) were also observed. ALP activity was inhibited by NaF (0.09-9 x 10(-8) M), with Vmax significantly decreased (P < 0.0001) at 0.09 M NaF. Alterations in the activity of these enzymes in the presence of fluoride may in part explain the decreased phosphorylation observed in DPP isolated from fluorotic dentine and may aid understanding of the altered matrix mediated mineralization patterns found during fluorosis.

Urine homogentisic acid and tyrosine: Simultaneous analysis by liquid chromatography tandem mass spectrometry

Alkaptonuria (AKU) is a rare debilitating autosomal recessive disorder of tyrosine metabolism. Deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid (HGA) which although excreted in gram quantities in the urine, is deposited as an ochronotic pigment in connective tissues, especially cartilage. Ochronosis leads to a severe, early-onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, Nitisinone, an inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of HGA, resulting in accumulation of the upstream pre-cursor, tyrosine. Using reverse phase LC-MS/MS, a method has been developed to simultaneously quantify urinary HGA and tyrosine. Using matrix-matched calibration standards, two product ion transitions were identified for each compound and their appropriate isotopically labelled internal standards. Validation was performed across the AKU and post-treatment concentrations expected. Intrabatch accuracy for acidified urine was 96-109% for tyrosine and 94-107% for HGA; interbatch accuracy (n=20 across ten assays) was 95-110% for tyrosine and 91-109% for HGA. Precision, both intra- and interbatch was <10% for tyrosine and <5% for HGA. Matrix effects observed with acidified urine (12% decrease, CV 5.6%) were normalised by the internal standard. Tyrosine and HGA were proved stable under various storage conditions and no carryover, was observed. Overall the method developed and validated shows good precision, accuracy and linearity appropriate for the monitoring of patients with AKU, pre and post-nitisinone therapy.

Potential problems with using deuterated internal standards for liquid chromatography-tandem mass spectrometry

We read with interest the recent article by Owen et al. on the importance of selecting the most suitable internal standard when validating a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. We are in agreement that any internal standard used should be thoroughly investigated during method development. Our recent experience in measuring 5-hydroxyindole acetic acid (5-HIAA) by LC-MS/MS using a d5 deuterated internal standard has highlighted a further issue not reported by Owen et al. It is considered best practice to use a deuterated analogue of the analyte being measured as they are proposed to have similar extraction recovery, ionization response in electrospray ionization mass spectrometry and the same chromatographic retention time. Moreover, these analogues should co-elute with the analyte to be quantified and contain enough mass increase to show a signal outside the natural mass distribution of the analyte. An important consideration with lyophilized internal standards is the solvent used for reconstitution and storage. Recently in our laboratory we changed from using a d2 (5-hydroxyindole-acetic-2,2-d2 acid) to a d5-labelled internal standard (5-hydroxyindole-4,6,7-d3-3-acetic-2,2-d2 acid) for the measurement of 5-HIAA. This was done to increase the signal outside the natural mass distribution of the analyte (i.e. the carbon 13 isotope of 5-HIAA). The d5-labelled internal standard (QMX Laboratories Ltd, Essex, UK) supplied contained no reconstitution or storage condition instructions. It was reconstituted and stored as a 10 g/L stock standard in 0.1 mol/L hydrochloric acid, as previously described by Perry et al. All stocks and dilutions were stored at 2208C. Serial dilution with 50:50 methanol:water allowed the d5 internal standard to be tuned (197.0.149.9) at a concentration of approximately 200 nmol/L and it was successfully introduced into our laboratory protocol. Within three months of introduction internal standard areas had dropped to 10% of the original peak area and external quality assessment data showed that our laboratory method had a positive bias. Evaluation of a parent ion scan revealed that the predominant m/z (mass to charge ratio) was 195.0 suggesting loss of two deuterium atoms (ion transition 195.0.147.0). Furthermore, a parent ion scan four weeks later revealed equal peak areas at both 194.0 (d2) and 195.0 (d3) parent masses. Personal communication with CDN isotopes suggested that the deuteriums at positions 4 and 6 were exchangeable. The loss of a third deuterium may be presumed to be at position 7. The total loss of three deuteriums resulted in the same parent mass as the previously used d2 5-HIAA. The mechanism for the loss of deuterium labels appears to be back-exchange of the label for hydrogen ions, present in the 0.1 mol/L hydrochloric acid used to reconstitute the original d5 5-HIAA internal standard. This phenomenon has not previously been reported in the literature for d5 5-HIAA internal standard. In our experience the d2 5-HIAA internal standard was more robust and was shown to be stable in 0.1 mol/L hydrochloric acid over an 18 month period. It is our recommendation that the solvent used to reconstitute deuterated internal standards be thoroughly investigated to avoid unnecessary loss in sensitivity. In this instance we recommend that d5 5-HIAA is reconstituted and stored in acetonitrile. Discussion with the supplier revealed the stability and storage data provided is only valid for the lyophilized product.

Acute fatal metabolic complications in alkaptonuria

Alkaptonuria (AKU) is a rare inherited metabolic disorder of tyrosine metabolism that results from a defect in an enzyme called homogentisate 1,2-dioxygenase. The result of this is that homogentisic acid (HGA) accumulates in the body. HGA is central to the pathophysiology of this disease and the consequences observed; these include spondyloarthropathy, rupture of ligaments/muscle/tendons, valvular heart disease including aortic stenosis and renal stones. While AKU is considered to be a chronic progressive disorder, it is clear from published case reports that fatal acute metabolic complications can also occur. These include oxidative haemolysis and methaemoglobinaemia. The exact mechanisms underlying the latter are not clear, but it is proposed that disordered metabolism within the red blood cell is responsible for favouring a pro-oxidant environment that leads to the life threatening complications observed. Herein the role of red blood cell in maintaining the redox state of the body is reviewed in the context of AKU. In addition previously reported therapeutic strategies are discussed, specifically with respect to why reported treatments had little therapeutic effect. The potential use of nitisinone for the management of patients suffering from the acute metabolic decompensation in AKU is proposed as an alternative strategy.