The Roles of Fibroblast Growth Factor (FGF)-23, α-Klotho and Furin Protease in Calcium and Phosphate Homeostasis : A Mini-Review

The roles of calcitonin, parathormone and calcitriol in the regulation of plasma calcium and phosphate are well-established. However, in autosomal-dominant hypophosphatemic rickety patients, studies have revealed normal plasma levels of calcium, associated with normal thyroid and parathyroid functions, but decreased levels of phosphate and calcitriol despite adequate reserves of vitamin D. Also, in tumoral calcinosis, persistent hyperphosphatemia with increased levels of 1,25(OH)₂D₃ have been observed. These studies indicate the involvement of factors other than the ones already known. The first decade of this century/millennium has led to the discovery of the involvement of fibroblast growth factor-23, furin protease and α-klotho in the homeostasis of calcium and phosphate, which is the subject of this mini-review.     

Association of Vitamin D Receptor (FokI and BsmI) Gene Polymorphism with Bone Mineral Density and Their Effect on 25-Hydroxyvitamin D Level in North Indian Postmenopausal Women with Osteoporosis

Osteoporosis is a systemic disease with a strong genetic component. Vitamin D receptor (VDR) has been suggested as a candidate gene for osteoporosis. Therefore the present study was aimed to investigate the pattern of allelic variants of VDR gene polymorphism (FokI and BsmI), its influence on vitamin D levels and bone mineral density (BMD) in North Indian postmenopausal women with osteoporosis for possible genetic association. 254 postmenopausal osteoporotic women and 254 postmenopausal non osteoporotic women were included in the study. VDR FokI and BsmI gene polymorphism gene were assessed by the PCR-RFLP method. Serum 25-hydroxyvitamin D was measured by the ELISA. BMD at the L₁–L₄ lumbar spine, hip, forearm and femoral neck was assessed by dual energy X-ray absorptiometry. The average BMD at spine and hip in postmenopausal women with bb and spine, hip, femoral neck and forearm with ff genotype had significantly low BMD. The frequency of ff genotype and f allele was significantly higher in postmenopausal osteoporotic women when compared with postmenopausal non osteoporotic women. However, no significant association was found between the genotypes and vitamin D levels. Our study reveals that VDR gene FokI and BsmI polymorphism is significantly associated with low bone mineral density. Therefore the ff genotype and f allele of VDR FokI gene may be used as an important risk factor for osteoporosis.     

1, 25-Dihydroxycholecalciferol (1,25-(OH)<Subscript>2</Subscript> D<Subscript>3</Subscript>) levels in osteoporosis

In the present study primary involution osteoporosis and vitamin D levels were studied in 60 subjects including thirty controls. The biochemical analysis of serum calcium, phosphorus, ALP, albumin and vitamin D₃ (1,25-(OH)₂ D₃) levels were significantly decreased in osteoporotic patients when compared to non-osteoporotic control group. There was a significant correlation of magnitude of sun exposure with 1,25-(OH)₂ D₃ levels. Similarly, vitamin D intake in the diet has a significant correlation with 1,25-(OH)₂ D₃ concentration. However, no correlation could be obtained between vitamin D₃ levels with increasing age. Among the biochemical markers serum calcium, phosphorus, ALP and albumin could not be correlated with 1,25-(OH)₂ D₃ levels.     

Status of Serum Calcium,Vitamin D and Parathyroid Hormone and Hematological Indices Among Lead Exposed Jewelry Workers in Dhaka,Bangladesh

Jewelry utilizes lead either directly or as a base metal. Costume jewelry requires lead before molding and plating the product with valuable metals. Therefore, such ornaments have a great potential to release heavy metals having health hazards. Also, jewelry makers engaged in preparing German silver, an alloy, apply lead in smelting, alloying, rolling and milling silver wires and pieces. The metal is taken up by blood, soft tissues and bone. The biological effects of lead are dependent upon the level and duration of exposure. Lead inhibits three enzymes of heme biosynthesis- δ-amino-levulinic-acid dehydratase (ALAD), coproporphyrin oxidase, and ferrochelatase, impairing heme synthesis and depressing serum level of erythropoietin resulting in decreased hemoglobin synthesis. Lead exposure also affects calcium metabolism and impair the synthesis of Calcitriol. In the present study, jewelry makers from Dhaka, Bangladesh, were shown to have significantly high levels of lead, protein, albumin, and parathormone in their blood, and significantly high amount of zinc-protoporphyrin and δ-amino-levulinic-acid in their urine. The control group, on the other hand showed significantly higher amounts of calcium (both total and ionized form) Vitamin D₃ and non-activated erythrocyte ALAD in their blood, along with hemoglobin. It might be due to inhibition of 1-α-hydroxylase enzyme in renal tubules. Lead causes nephro-toxicity and inhibits 1-α- hydroxylase enzyme leading to decreased calcitriol synthesis resulting in impaired calcium absorption across gastro-intestinal tract and renal tubules. Low Vitamin D₃ and significantly increased Parathyroid hormone (PTH) in study group has been found.     

Method evaluation study of a new generation of vitamin D assays

Introduction

Recently several diagnostic manufacturers have launched new 25-hydroxy-vitamin D (25[OH]D) assays, which are aligned to the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRM) (NIST, Gaithersburg, Maryland). The aim of this study was to compare the performance of one liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, one enzyme linked immunosorbent assay (ELISA), and one recalibrated and previous version of a chemiluminescence immunoassay (CLIA).

Material and methods

Serum-aliquots of 198 patient samples from routine 25(OH)D analysis were measured by the ClinMass® LC-MS/MS Complete Kit (RECIPE Chemicals + Instruments GmbH, Munich, Germany), the ORGENTEC 25(OH)D₃/D₂ ELISA (ORGENTEC Diagnostika GmbH, Mainz, Germany), the recalibrated Immunodiagnostic Systems (IDS)-iSYS 25(OH)D^S and the previous used IDS-iSYS 25(OH)D CLIA (Immunodiagnostic Systems Ltd, Boldon, United Kingdom). Bland-Altman and Deming regression analyses were calculated for methods comparison of all tested 25(OH)D assays. The LC-MS/MS method was defined as the reference method. Within-run and between-run precision measurements were performed for all methods with three different concentration levels.

Results

Compared to the LC-MS/MS method, the new IDS-iSYS 25(OH)D^S and ORGENTEC 25(OH)D₃/D₂ assay demonstrated mean relative biases of 16.3% and 17.8%. The IDS-iSYS 25(OH)D assay showed the lowest mean bias of 1.5%. Deming regression analyses of the recalibrated IDS-iSYS 25(OH)D^S and the ORGENTEC 25(OH)D₃/D₂ assay showed proportional differences, when compared to the reference method. All assays showed a within-run and between-run imprecision of ≤ 20% at each of the evaluated concentration levels.

Conclusions

The evaluated standardized immunoassays and LC-MS/MS are useful methods for measuring 25(OH)D serum-levels in clinical laboratories.Key words: vitamin D, immunoassays, liquid chromatography-tandem mass spectrometry, reference standards, quality improvement     

Status of Vitamin D Receptor Gene Polymorphism and 25-Hydroxy Vitamin D Deficiency with Essential Hypertension

Essential hypertension (EH) is a multifactorial and complex disease with high rate of incidence and associated co-morbidities. Previous studies do not provide unanimous results for the risk of hypertension and association with Fok I genotype frequency and serum vitamin D levels. Hence, this study was undertaken to determine the status of Fok I vitamin D receptor (VDR) gene polymorphism along with vitamin D levels and blood pressure in patients with EH. Four hundred (200 controls and 200 cases of essential hypertension) participants from general Indian population were enrolled in this study. Peripheral blood samples were collected for genotyping Fok I-VDR gene polymorphism using PCR–RFLP method whereas 25-OH vitamin D levels in serum were quantified using high performance liquid chromatography (HPLC). Significantly reduced 25-OH vitamin D levels were observed in patients with EH (24.04 ± 8.62 vs 50.46 ± 15.46) compared to control subjects (p = 0.0001). Homozygous recessive genotype ‘ff’ frequency was increased by 8.06 fold (CI: 3.71–17.47, p = 0.0001) in patients with EH compared to dominant ‘FF’ genotype frequency. In conclusion, recessive ‘ff’ genotype frequency correlates with reduced serum vitamin D levels and results in significantly increased systolic and diastolic blood pressures leading to predisposition of EH.     

Education and communication is the key for the successful management of vitamin D test requesting

Introduction

Pre-preanalytical and post-postanalytical phases are steps where the laboratory professional may play a crucial role. Measuring the serum circulating 25 hydroxyvitamin D level (25(OH)D) is recommended to evaluate vitamin D status in patients at risk for vitamin D deficiency while 1,25 hydroxyvitamin D (1,25(OH)₂D) is only recommended to monitor several particular conditions (chronic kidney disease, hereditary phosphate-losing disorders, and some other) clearly defined by the current clinical guidelines of Endocrine Society.
Our research hypothesis was that through education and communication through comments in the Laboratory Information System (LIS), we could improve appropriateness in the request vitamin D tests.

Materials and methods

A retrospective observational cross-sectional study was conducted from January 2005 to December 2014. Each 1,25(OH)₂D request was reviewed individually by a member of the laboratory staff. Starting in November 2011, each inappropriate 1,25(OH)₂D request was registered in LIS and 25(OH)D was measured instead of 1,25(OH)₂D. We counted the overall number of 1,25(OH)₂D requests and the number of inappropriate requests which then were marked with a comment.

Results

The request of 25(OH)D increased along years. However, 1,25(OH)₂D requests increased until 2012 when demand began to diminish.

Conclusions

Education and communication through comments in the LIS, corrected the inappropriate request of 1,25(OH)₂D and promoted the use of 25(OH)D to study vitamin D deficiency.Key words: clinical laboratory services, utilization, vitamin D, communication, education     

Predisposition of genetic polymorphism with the risk of urolithiasis

Urolithiasis is a relevant clinical problem with a subsequent burden for health system. The aim of this review is to provide recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for kidney stone disease and to predict treatment response. Population case-control studies are useful both as an alternative and an adjunct as compared to family studies. These involve either whole genome scanning or candidate gene approaches. While whole genome scanning is likely to be widely used in future, at present, candidate gene studies are more feasible. When performing candidate gene case-control studies factors such as study design, methods for recruitment of case and controls, selection of candidate genes, functional significance of polymorphisms chosen for study and statistical analysis require close attention to ensure that only genuine associations are detected. Some of the significant genes that play role in stone formation include calcitonin receptor gene (CTR), vitamin D receptor (VDR), Urokinase, Interleukin, (IL-1β, IL-Ra), E-Cadherin, Androgen & oestrogen receptor gene, vascular endothelial growth factor (VEGF) and Arginine p21. In our case-control study we studied CTR, VDR, Urokinase, IL-1β(−511 and +3954), IL-Ra from north India and predict that VDR, IL-β (-511) and IL-1Ra gene may be used as a possible genetic marker for earlier detection in patients who are at risk for calcium oxalate stone disease. Further, linkage disequilibrium and haplotype structure of a certain candidate gene is important for association analysis. When a certain polymorphic allele has been found to be associated with disease, it is further explained on basis of LD and haplotype structure by one or more other alleles. Once it is determined which haplotype carries the risk allele, by means of molecular biological functional analyses, the variants on that haplotype allele truly causing the effect can be determined.     

Effect of folic acid and vitamin B<Subscript>12</Subscript> administration on phenytoin induced toxicity in rats

Folic acid and vitamin B₁₂ are very important vitamins needed for normal cellular metabolic activities. The effects of folic acid and vitamin B₁₂ on liver integrity of growing Wistar albino rats following therapeutic dose of phenytoin administration were investigated. The activities of serum AST, ALT, ALP were investigated. Serum total protein level and lipid profile were also measured as indices of biochemical changes. The ingestion of phenytoin alone in rats significantly reduced serum protein while AST, ALT activities incresed as compared to the control (P<0.05). Supplementation of phenytoin with oral administration of 70microgram/kg body wt of folic acid resulted in a significant reversal in serum total protein and suppression in serum AST and ALT activities. Vitamin B₁₂ supplementation did not afford any significant protection against the effect of phenytoin ingestion but rather phenytoin toxicity was exacerbated in this study. However, the combined effects of vitamin B₁₂ and folic acid ameliorated the effects of phenytoin on serum enzymes of experimental rats. The effect of combination of phenytoin with folic acid or folic acid and vitamin B₁₂ is an interesting finding. Supplementation of phenytoin with folic acid or combination of these vitamins may be recommended for the purpose of ameliorating the adverse biochemical changes which are associated with phenytoin therapy. Further work is ongoing to help elucidate the effects of phenytoin and these vitamins on oxidative stress inducing mechanism.     

The Role of 25-Hydroxy Vitamin D Deficiency in Iron Deficient Children of North India

Extensive data from animal and human studies indicate a role of vitamin D in erythropoiesis. Iron and vitamin D deficiencies are implicated with adverse health effects in children even if they are asymptomatic. The potential relationship between the two remains poorly understood. A cross-sectional study was performed in the period from 1st May 2012 through 30th April 2013 and subjects were classified into vitamin D deficiency (VDD), vitamin D insufficiency (VDI) and vitamin D sufficiency (VDS) groups according to their 25(OH) D levels. A total of 263 children were included in the analysis. Anaemia was present in 66 % of 25(OH) D deficient subjects compared with 35 % in vitamin D sufficient individuals (p < 0.0001). The association of breast feeding and development of VDD was also significant (p < 0.05). Serum levels of 25(OH) D were found lower in female sex and if the analysis was performed in the winter/spring season. Physicians should therefore assess vitamin D levels in all anaemic children and ensure adequate supplementation to prevent deficiencies.     

Role of Vitamin D Supplementation in Hypertension

Role of Vitamin D supplementation was studied in patients with hypertension. One hundred hypertensive patients (group I) were given conventional antihypertensive drugs while another 100 patients (group II), in addition, were supplemented with Vitamin D₃ (33,000 IU, after every 2 weeks, for 3 months). Besides diastolic and systolic blood pressure, serum calcium, phosphorous, alkaline phosphatase, albumin, albumin-corrected calcium, and 24 h urinary creatinine levels were estimated in both the groups before the start of treatment and after 3 months. Vitamin D supplementation showed a more significant decrease in systolic blood pressure. This group also showed a significant increase in serum calcium as well as albumin-corrected calcium with a decrease in phosphorous. Results of the study confirm that Vitamin D supplementation has a role in reducing blood pressure in hypertensive patients and that it should be supplemented with the antihypertensive drugs. More extensive studies with a larger group, to draw a definite conclusion, are in progress.     

In vitro study of vitamin B6 status in cancer breast patients

There is ample evidence that vitamin status of an individual can have a profound influence on the resistance of Cancer. Pyridoxine is one of the water soluble vitamins playing an important role in various metabolic reactions. Aspartate aminotransferase and Alanine aminotransferase are pyridoxal phosphate requiring enzymes and their activities in erythrocytes have been used to assess the state of vitamin B₆ nutrition in man. We report here the erythrocyte transaminase levels in controls and patients with early and advanced cancer of breast. Thein vitro study of vitamin B₆ shows a marked difference in the per cent stimulation in patients as compared to the controls on addition of pyridoxal phosphate. The difference indicates a subclinical deficiency of vitamin B₆. The possibility exists that the rapid progression of the disease could be due to lack of host resistance caused by the deficiency of vitamin B₆. The possibility of an effective treatment response after enhancing the vitamin B₆ status is being looked into.     

Secondary electron emission from oxide-coated cathodes

The secondary electron emission from alkaline-earth oxide-coated cathodes has been investigated under both continuous and pulsed bombardment. Various factors affecting the yield, such as dependence upon primary voltage, collecting voltage, temperature, time, and angle of incidence, are noted, and the present state of the theory is discussed.Experiments have been performed with three types of apparatus. Yield vs. Energy data reveal values of δ of 4–7 at room temperature, with a more or less flat maximum at approximately 1,000 volts primary energy.The yield increases with temperature in an exponential manner, and plots of log Δδ (i.e. δ_K° ? δ_300°K) vs. 1/T give straight lines. Values of Q₁ between 0.9 – 1.5 eV. are generally indicated, and from extrapolation of these curves, yields exceeding 100 at 850° C. are deduced. The secondary emission depends upon the degree of activation, and increases with enhancement of the thermionic emission characteristics. Short-time effects such as growth or decay of secondary current after the onset of primary bombardment or persistence after the cessation of bombardment have not been observed, and values of yield obtained by pulsed methods are in accord with those obtained under D.C. conditions. Tail phenomena reported by J. B. Johnson and interpreted as “enhanced thermionic emission” from oxidecoated cathodes become manifest only under experimental conditions characterized by certain space-charge effects, and have been effectively simulated by bombarding a tantalum target adjacent to an electron-emitting tungsten filament. Various measurements of the energy distribution of secondary electrons as a function of primary voltage and temperature have been obtained. It was observed that the average energy of the secondary electrons decreases with temperature at a rate which more than compensates for the increase in the number of secondaries emitted per incident primary. The mechanism of the observed dependence of yield upon temperature is not well understood. Various alternative explanations are discussed and, in the light of the present state of our knowledge, regarded as untenable.     

A modified PID controller (PIIσβD)

The purpose of this study is to modify the traditional PID controller in order to improve its performance (stability and tracking) by changing the length of integration interval. The performance of the traditional PID controller was improved by changing the length of integration interval to make the most of the returns of the PID and PI_σD controllers. The asymptotic stability domain, in terms of the feedback gains, is derived for systems of second order using the modified controller which will be identified as PII_σ^βD. Comparing this controller with the traditional PID controller and PI_σD controller proposed in [1], it proves that it is more accurate and more stable. For illustration and comparison, two examples have been simulated to evaluate the performance of the modified controller. All simulation results indicate that the modified controller is better than the traditional PID controller and the PI_σD controller from the accuracy and stability point of view.     

Tracking structural evolution: operando regenerative CeOx/Bi interface structure for high-performance CO2 electroreduction

Unveiling the structural evolution and working mechanism of catalysts under realistic operating conditions is crucial for the design of efficient electrocatalysts for CO₂ electroreduction, yet remains highly challenging. Here, by virtue of operando structural measurements at multiscale levels, it is identified under CO₂ electroreduction conditions that an as-prepared CeO₂/BiOCl precatalyst gradually evolves into CeO_x/Bi interface structure with enriched Ce³⁺ species, which serves as the real catalytically active phase. The derived CeO_x/Bi interface structure compared to pure Bi counterpart delivers substantially enhanced performance with a formate Faradaic efficiency approaching 90% for 24 hours in a wide potential window. The formate Faradaic efficiency can be further increased by using isotope D₂O instead of H₂O. Density functional theory calculations suggest that the regenerative CeO_x/Bi interfacial sites can not only promote water activation to increase local ^*H species for CO₂ protonation appropriately, but also stabilize the key intermediate ^*OCHO in formate pathway.     

Association between plasma homocysteine and riboflavin status in Acute Lymphoblastic Leukemia in children

Remethylation of homocysteine to methionine is dependent on an adequate supply of one or more of the B vitamins like folate, vitamin B₁₂ and vitamin B₆. Plasma total homocysteine (tHcy) is also influenced by genetic factors such as polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene. MTHFR is a flavo enzyme and a key player in folate metabolism and changes in its activity could modify the susceptibility to Acute Lymphoblastic Leukemia (ALL). In this case — control study we have examined the effect of riboflavin status as measured by erythrocyte glutathione reductase activation coefficient (EGRAC) on homocysteine levels along with vitamin B₁₂ and folate in pediatric ALL. Folate and B₁₂ levels were significantly lower among cases as compared to controls while EGRAC and tHcy did not differ significantly among the groups. The multivariate regression analysis revealed that in the ALL group EGRAC significantly influences tHcy levels suggesting that riboflavin availability may be a predictor of tHcy levels in patients with ALL. This finding may have implications for tHcy lowering therapy.     

Serum Vitamin D Levels in Indian Patients with Multiple Sclerosis

Low serum vitamin D level has an increased association with risk of multiple sclerosis (MS).There has been no published data on the levels of this vitamin in Indian population with MS. Hence we decided to undertake this study to document if there is evidence of vitamin D deficiency in patients with MS in our population. 26 patients with diagnosis of MS by modified Mc Donald’s criteria were enrolled in this study. Serum vitamin D (1,25 hydroxy) levels were measured by electro-chemiluminescence in our biochemistry lab. An age-matched control group of 202 patients who did not have a diagnosis of MS were included. In our study group 76.9 % had vitamin D level less than 20 ng/ml compared to 65.5 % of control group (p value of 0.019). Our study revealed a trend towards low vitamin D values in Indian MS patients.     

Involvement of Depressive Catecholamines as Thrombosis Risk/Inflammatory Markers in Non-Smoker,Non-Obese Congestive Heart Failure,Linked to Increased Epidermal Growth Factor-Receptor (EGF-R) Production

The EGF-R, also known as HER-1 or erbB-1 (EGF-R/HER-1/erbB-1), is a member of the human epithelial receptor tyrosine kinase family. sEGF-R is considered to play a role in cardiac (patho)physiology. We aimed to investigate whether soluble EGF-R is increased in congestive heart failure (CHF) patients and if related to disease severity. Soluble EGF-R, vitamin D, parathyroid hormone (PTH) was studied, and being evaluated in relation to Ca²⁺, lipids, hsCRP, fibrinogen, serotonin, norepinepherine (NE). The study compared non-smoker, non-obese male CHF patients (n = 50) with age and gender-matched essential hypertension (HTN) patients (n = 20). Moreover, comparison with healthy control volunteers (n = 20) were employed. EGF-R/HER-1/erbB-1 was higher (P = 0.013) in 50 CHF male patients mean 12 ± 0.7 fmol/ml, than in 20 HTN, 9.25 ± 0.6 fmol/ml or in 20 controls, 6.25 ± 1 fmol/ml. Serum EGF-R levels correlated positively with hsCRP and NE, and were highest among CVD patients (n = 70) as well as negatively with vitamin D and HDL-C. EGF-R/HER-1/erbB-1 levels are increased in HTN and more in CHF patients. This study confirms a strong association between catecholamines as well as EGF-R/HER-1/erbB-1 levels with PTH and low vitamin D levels, being related to hyperlipidemia and inflammation (hsCRP and fibrinogen) in CVD. Moreover, contributing to the complex process of the inflammatory component of atherosclerosis in hypertensive patients that leads eventually to CHF.     

Assessment of Vitamin D status In Patients of Chronic Low Back Pain of Unknown Etiology

Low back pain is very disabling and dispiriting because of the physical impediment it causes and its psychological effects. Innumerable factors have been implicated in its etiology. In spite of improvements in diagnostic modalities, a considerable number of such cases fall in the ambiguous zone of unknown etiology or ‘idiopathic.’Early diagnosis of low back pain will allow effective prevention and treatment to be offered. This study was conducted to assess the contribution of vitamin D levels and other biochemical factors to chronic low back pain in such cases. All patients attending the orthopedics OPD for low back pain in whom a precise anatomical cause could not be localized, were prospectively enrolled in this study. We measured serum levels of glucose, calcium, phosphorus, uric acid, rheumatoid factor, C reactive protein, alkaline phosphatase, total protein, albumin and 25 (OH) D concentrations in 200 cases and 200 control samples. The patients showed significantly lower vitamin D levels compared to controls with p value < 0.0001. The maximum number of low back pain patients were in the age group of 31–50 years (42 %).The average BMI was 23.27 ± 5.17 kg/sq m, 73 % of total patient population were females and 27 % were known case of type 2 diabetes mellitus. Calcium, alkaline phosphatase, was positively correlated with vitamin D and glucose showed a negative correlation with vitamin D in the patient population. The problem of low back pain provides a challenge to health care providers. The problem in developing countries is compounded by ignorance to report for early treatment and occupational compulsions in rural areas and sedentary lifestyle in urban youth. The authors strongly recommend early frequent screening for vitamin D along with glucose, protein, albumin, calcium, phosphorus, CRP as part of general health checkup for non-specific body pain, especially low back pain.