Study on Urinary Candidate Metabolome for the Early Detection of Breast Cancer

A metabolomic study for determination of certain urinary metabolomes, 1-methyladenosine (1-MA), 1-methylguanosine (1-MG), and 8-hydroxy-2′ deoxyguanosine (8-OHdG) in urine specimens of breast cancer patients. The accuracy of these metabolites and their combined score with cancer antigen 15-3 (CA15-3) was developed to improve the early detection of breast cancer. This study recruited 52 healthy individuals, 47 benign breast tumors, and 167 malignant breast tumor patients. Urine samples were handled to adjust the creatinine concentrations to 8 mg/dL (0.7 mmol/L) and analyzed using GC–MS to detect and quantify the selected urinary metabolomes in urine samples of all participants. The accuracy of individual urinary metabolomes and their combination with CA15-3 were evaluated using multivariate statistical analysis. The cutoff value of CA15-3 was 32.5 U/mL. Cutoff values of 1-MA, 1-MG, and 8-OHdG were 2.19, 2.1, and 7.3 µmol/mmol creatinine, respectively. The concentrations of 1-MA, 1-MG, and 8-OHdG were significantly higher in breast cancer patients, especially in the early-stage. The combination of three urinary metabolomes with CA15-3 improves the diagnostic sensitivity of breast cancer. For the combined score, the area under the curve (AUC) value of combined score ranged from 0.820 to 0.950, with high accuracy, ranged from 77.0 to 95.5%. The most significant AUC (0.973), sensitivity (90.1%), selectivity (94.0%) was recorded at comparing the healthy control with the early-stage of malignant breast cancer. In conclusion, the combination of three urinary metabolomes with serum CA15-3 improves the diagnostic sensitivity of breast cancer.     

Metabolic Bone Profile of Healthy Adult North Indian Population from Chandigarh Urban Bone Epidemiological Study (CUBES)

We aimed to estimate metabolic bone profile in a large cohort of healthy, adult Indian population to generate reference standards of serum calcium, phosphate and alkaline phosphatase (ALP), 25 (OH) Vitamin D and iPTH, and also to find out the prevalence of Vitamin D deficiency in healthy population. Apparently healthy people in the age group of 20–80 years, residing in the union territory of Chandigarh were chosen. Fasting samples for serum calcium, phosphate, albumin, alkaline phosphatase (ALP), 25 (OH) D and iPTH were collected and were processed on the same day. We recruited 930 healthy subjects from different subsectors of Chandigarh. Final analysis was done for 915 subjects. Out of this, 530 (58%) were women and 385 (42%) were men. The study participants were divided into two groups, less than and more than 50 years for the men and pre and post-menopausal for the women. The serum calcium, phosphate, ALP and iPTH were significantly higher in the post-menopausal women compared to the pre-menopausal women. The median plasma 25 (OH) D in men and women was 12.5 ng/mL and 14.3 ng/mL, respectively. 25 (OH) D deficiency was seen in 65.4% of individuals. 25 (OH) D levels co-related negatively with iPTH levels (r = − 0.4, p < 0.0001), and showed an increasing trend with age. We have thus presented metabolic bone profile of healthy, adult north Indian population. These reference values can be used for diagnosis and monitoring of various MBDs. Vitamin D deficiency is still rampant in our population in spite of increasing awareness.     

Detection and correction of incomplete duplicate 24-hour urine collections – theory and practical evidence

IntroductionThe intraindividual variability in urinary creatinine excretion is notoriously large. The aims of this study were to investigate the variability of duplicate consecutive 24-hour urinary creatinine excretions in patients and to develop a model for the detection and correction of discrepant creatinine excretions.Materials and methodsA group of 270 patients (82 men and 188 women) were included in the study. We collected the following data: urinary 24-hour volumes (volumetric/gravimetric) and urinary creatinine concentrations (Jaffé/enzymatic) on both collection days. We performed specific calculations to detect discrepant creatinine excretions.ResultsIn 60 patients (22%) discrepant collections were found. Among the remaining 78%, 22% of the patients collected very accurately (almost identical urinary creatinine excretions). In this subgroup the volume ratios and the creatinine concentration ratios behave inversely as in a dilution curve. A theoretical model and six collection scenarios were developed to detect, interpret and correct discrepant collections. Practical examples are given to illustrate the use of the model in successful correction of creatinine and other analytes for under- or overcollection.ConclusionsWe conclude that missed or overcollected urine volumes are the largest source of variation in creatinine excretion. Discrepancies in consecutive duplicate 24-hour creatinine excretions can be detected and corrected with specific calculations by means of the presented model. The effectiveness of these corrections is demonstrated with examples from daily practice. These calculations can be easily automated.     

Women in sports: the applicability of common national reference intervals for inflammatory and coagulation biomarkers (HemSter Study)

IntroductionIntensive physical activity causes functional and metabolic changes in the athlete’s organism. The study aimed to verify the common national available reference intervals (RIs) for common inflammatory and screening coagulation tests in a population of healthy young female athletes.Materials and methodsOne hundred and twenty-one female athletes (age range: 16–34), from various sports disciplines (water polo, handball, volleyball, football, basketball), were included in the study. All participants completed the international physical activity short-form questionnaire. Blood samples were collected between 8–10 am, after an overnight fast, before any physical activity. Reference intervals were determined according to Clinical & Laboratory Standards Institute EP28-A3C Guidelines.ResultsCalculated RIs for white blood cell count (WBC), prothrombin time (PT), and activated partial thromboplastin time (APTT) ratio were in accordance with the common national RIs. Calculated RI for C-reactive protein (CRP) was lower (< 2.9 mg/L) than the proposed cut-off for a healthy population (< 5.0 mg/L). Reference interval for fibrinogen was higher (1.9–4.4 g/L), than the available RIs (1.8–3.5 g/L). D-dimer cut-off value was set at 852 µg/L fibrinogen equivalent units (FEU), higher than the proposed 500 µg/L FEU for venous thromboembolism (VTE) exclusion.ConclusionsThe applicability of the available RIs for WBC count, PT, and APTT-ratio was confirmed. However, RIs for CRP and fibrinogen differed significantly than the available common national RIs for the healthy non-athletes’ population. A higher cut-off for D-dimers should be extensively verified before implementation for VTE diagnosis exclusion in a group of healthy young female athletes.     

Acute Renal Failure in Liver Transplant Patients: Indian Study

The acute renal failure is the frequent medical complication observed in liver transplant patients. The objective of this study was to determine the cause of acute renal failure in post liver transplant patients. A total of 70 patients who underwent (cadaveric 52, live 18) liver transplantation were categorized based on clinical presentation into two groups, namely hepatorenal failure (HRF, n = 29), and Hepatic failure (HF, n = 41). All the patients after the liver transplant had received tacrolimus, mycophenolate and steroids. We analyzed the modification of diet in renal disease, (MDRD) serum urea, creatinine and albumin before and after 5th and 30th day of liver transplant and data was categorized into survivors and non-survivors group. In HRF survivor group, serum creatinine, and urea levels were high and, albumin, MDRD were low in pre- transplant and reached to normal levels on 30th day of post transplant, and 79.3 % of patients in this group showed resumption of normal kidney function. On the contrary in HRF nonsurvivor group, we did not observed any significant difference and 20.7 % of patients showed irreversible changes after the liver transplant. In HF survivor group, 82.9 % of liver failure patients did not show any deviation in serum creatinine, urea, albumin and MDRD, whereas in HF non survivor group, 17.1 % of liver failure patients who had HCV positive before the transplant developed acute renal failure. The levels of creatinine, urea, albumin and MDRD were normal before the transplant and on day 30th, the levels of albumin and MDRD were significantly low whereas serum urea, creatinine levels were high. In conclusion, based on these observations, an diagnosis and treatment of Acute renal failure is important among the liver transplantation cases in the early postoperative period.     

Urinary Glycosaminoglycan Estimation as a Routine Clinical Service

Mucopolysaccharidoses, a group of inherited disorders are associated with defects in glycosaminoglycan metabolism. Thus, assessment of urinary glycosaminoglycan is used as a screening test for mucopolysaccharidoses. The detection methods range from qualitative spot tests to quantification using metachromatic dyes. In our laboratory we optimized a spectrophotometric quantitative method using a metachromatic dye, dimethylmethylene blue. Heparan sulfate was used for quantification. The glycosaminoglycan–dye complex showed a marked shift in color with increase in concentration. The color complex was quantified at 520 nm. The method was linear from 10–89 mg/L. An age matched normal range was obtained in 177 healthy individuals, grouped in 8 different age groups from neonates to adults. Urinary glycosaminoglycan concentration varied distinctly amongst the study population wherein the lowest range in healthy neonates was more than 3 times the upper limit of healthy adults. Urine samples from 10 patients with mucopolysaccharidoses were also included in the study for clinical validation. The method qualified both analytical and clinical validation and was found to be simple, robust and ideal to be offered as a screening test for mucopplysaccharidoses in a routine clinical chemistry laboratory.     

The Plasma DNA Concentration as a Potential Breast Cancer Screening Marker

Increased levels of plasma DNA have frequently been noticed in the blood plasma of cancer patients. The possibility of using plasma DNA level as the indicator of tumor stage in breast cancer was investigated in plasma samples obtained from 100 breast cancer patients and 100 healthy women who were included as controls. Circulatory plasma free DNA was extracted from plasma samples and quantified by fluorometer. The median concentration of plasma DNA in the plasma samples from breast cancer patients classified by TNM staging system as stage I, II, III, IV and breast surgical patients were 0.5, 235, 422, 1,280 and 0.5 ng/ml, respectively. The level of plasma DNA in the stage II- IV group was significantly higher than those in the surgical group with breast cancer and control group (P value < 0.001). The plasma DNA concentration in stage II, III and IV of breast cancer were higher when compared with healthy group. These tumor size, TNM stage and metastasis were significantly correlated with plasma DNA. The cut point of 120 ng/ml was early screening and treatment follow up breast cancer.     

Correlation of Microalbuminuria with Estimated GFR (eGFR) by Cockcroft–Gault and MDRD Formula in Type 2 Diabetics and Hypertensives

Increase in urine albumin excretion rate (AER) precede a fall in glomerular filtration rate in patients developing diabetic chronic kidney disease (CKD). Our results have shown that 7 (50 %) of diabetic and hypertensive individuals with decreased GFR do not have increased AER. In this cross-sectional study, we measured AER of 75 patients with type 2 diabetes and hypertension by immunoturbidimetric method. We correlated the results with eGFR values obtained by Cockcroft–Gault and MDRD method. The method used was not a compensated method. We measured serum creatinine by modified Jaffe’s kinetic method in autoanalyzer XL-600. Analysis of data showed positive correlation between eGFR and microalbuminuria by both the methods with eGFR <60 mL/min/1.73 m². Pearson’s correlation co-efficient (r) was 0.9 (p = 0.0001) by Cockcroft–Gault formula and 0.69 (p = 0.0063) by MDRD formula. Our results concluded that there was positive correlation between AER and eGFR <60 mL/min/1.73 m². We have recognized that these two parameters provide a complimentary benefit in management of cases with CKD.     

Diagnosis of Major Organic Acidurias in Children: Two Years Experience at a Tertiary Care Centre

Organic acid disorders are inherited metabolic disorders in which organic acids accumulate in tissues and biological fluids of affected individuals. Classical organic acidurias include methylmalonic aciduria, propionic aciduria, isovaleric aciduria and maple syrup urine disease (MSUD). They are considered the most frequent metabolic disorders among severely ill children. Patients frequently present with acute symptoms early in life. 420 cases clinically suspected to have organic aciduria, with upper age limit of 12 years for a 2-year period (January 2007–December 2008) were enrolled into this study. Metabolic acidosis and neurological symptoms were the most common signs. Screening tests and thin layer chromatography were done for detection of organic acidurias. Identification and quantitation of organic acids in urine and quantification of amino acids in blood were done by high performance liquid chromatography. Out of 420 patients, 45 patients (10.7%) were found to have organic acidurias. 15 cases of methylmalonic aciduria, 16 cases of propionic aciduria, 13 cases of MSUD, and one case of isovaleric aciduria were diagnosed. Results demonstrate the importance of testing for organic acidurias. Since organic aciduria may cause irreversible brain damage if not treated, we recommend selective screening amongst severely ill children despite implied extra costs.     

Effect of Oestrogen on Altering the Serum and Urinary Levels of Calcium,Phosphate and Magnesium in Hysterectomised Women Compared to Natural Menopausal South Indian Women: A Case Control Study

Given the paucity of studies conducted to know the effect of suddenness and earlier onset of endocrinological changes associated with hysterectomy, on the serum and urinary levels of calcium, magnesium and phosphate the present study was conducted to compare the levels of calcium, magnesium and phosphate in serum and urine of hysterectomised and natural menopausal south Indian women. This is a cross-sectional observational study. The study included three groups of 30 healthy premenopausal, 30 early surgical menopausal and 30 natural post menopausal women. Women suffering from any endocrine disease were excluded. Analysis was performed in serum and urine sample. The levels of calcium, magnesium and phosphate in serum and calcium/creatinine, magnesium/creatinine and phosphate/creatinine ratio were estimated in urine by spectrophotometric method. Hysterectomised women (serum calcium: 8.7 ± 0.09 mg/dl; urine calcium/creatinine: 0.16 ± 0.02) have significantly low serum calcium (p < 0.001) and high urinary calcium/creatinine (p = 0.002) ratio and post menopausal women (serum magnesium: 2.1 ± 0.03; serum phosphate: 4.4 ± 0.16; urinary calcium/creatinine: 0.17 ± 0.02; urinary magnesium/creatinine: 0.09 ± 0.01) have significantly high serum magnesium (p = 0.016), serum phosphate (p = 0.043) and high urinary calcium/creatinine (p = 0.002), magnesium/creatinine ratio (p = 0.025) compared to healthy pre menopausal women. Post menopausal women (serum calcium: 9.1 ± 0.08) have significantly high serum calcium and phosphate compared to hysterectomised women (serum phosphate: 3.93 ± 0.11). Hysterectomised women have significantly low serum calcium, oestrogen and high urinary calcium/creatinine ratio compared to healthy premenopausal women and low serum calcium and low serum phosphate compared to natural postmenopausal women. Natural postmenopausal women had low serum oestrogen and high serum magnesium, serum phosphate, urinary calcium creatinine ratio and urinary magnesium creatinine ratio compared to healthy premenopausal women.     

A Comparative Study of Clinical Utility of Spot Urine Samples with 24-h Urine Albumin Excretion for Screening of Microalbuminuria in Type 2 Diabetic Patients

Twenty-four hour urinary albumin excretion (UAE) is considered as gold standard method for albuminuria measurement, but collection of 24-h urine is inconvenient. The aim of present study was to evaluate whether albumin: creatinine ratio (ACR) and urinary albumin concentration (UAC) in different spot urine samples correlate or not with 24-h UAE for screening of microalbuminuria in type 2 diabetic patients. We collected first morning void (FMV), random urine sample (RUS) and 24-h urine, separately on consecutive days from 104 type 2 diabetic patients. ACR and UAC in each spot urine sample compared with 24-h UAE with regard to Pearson correlation coefficient. Pearson’s correlation of albumin: creatinine ratio (ACR) with 24-h UAE was (r = 0.802 and 0.623) in first morning void (FMV) and random urine sample (RUS), respectively. Pearson’s correlation coefficient of urinary albumin concentration (UAC) compared with 24-h UAE was (r = 0.943 and 0.920), in FMV and RUS, respectively, P < 0.01. Results revealed that values in first morning void (FMV) were better correlated with 24-h urinary albumin excretion (UAE), than the values in random urine sample (RUS). We conclude that the first morning void (FMV) may be able to replace 24-h urine collection, preferably urinary albumin concentration (UAC) in the initial screening of microalbuminuria in diabetic patients.     

Method Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry for Angiotensin-II in Human Plasma: Application to Study Interaction Between Atorvastatin & Olmesartan Drug Combination

Simple and sensitive Liquid Chromatography-Tandem Mass Spectrometry (LCMS/MS) method was developed and validated, then was implicated on hypertensive human subjects to study drug interaction of atorvastatin (ATVS) and Olmesartan (OLM) on status of Angiotensin-II (ANG-II). The ANG-II in plasma was extracted with 5 mL methanol containing 5 % formic acid through C₁₈ (cartridges) liquid–liquid extraction, dried and reconstituted with 1 mL of 16 % acetonitrile in 0.1 % formic acid in water. The chromatographic separation of ANG-II with a Agilent technology 6410 Triple quadrupole was carried multiple reaction monitoring scan mode with a Agilent 1290 Infinity LC system for UHPLC. The sample were separated on a (Thermo Scientific) Hy-Purity advance (50 × 4.6 mm, 5 μm) using Mobile Phase A: 16 % acetonitrile in 0.1 % formic acid in water and Mobile Phase B: 0.1 % formic acid in methanol at a flow rate of 0.3 mL/min, performed at ambient temperature. The mobile phase gradient of 16 % acetonitrile in water was linearly increased to 38 % acetonitrile over 10 min and subsequently the mobile-phase was increased to 100 % acetonitrile over 15 min. The developed method was validated for specificity, accuracy, precision, stability, linearity, sensitivity and recovery. The method was linear between peak area ratio of standard and internal standard over the range of 50–800 ng/mL. The method was successfully applied for the drug interaction study revealed levels of ANG-II were significantly higher in ATVS + OLM treatment condition as compared to individual treatment of OLM. This reflects the reason of low effectiveness of ATVS + OLM in combination instead of synergistic activity.

Electronic supplementary material

The online version of this article (doi:10.1007/s12291-014-0457-x) contains supplementary material, which is available to authorized users.     

Study of the Concentration of Trace Elements Fe,Zn, Cu,Se and Their Correlation in Maternal Serum,Cord Serum and Colostrums

A study of iron, zinc, copper and selenium concentration levels was carried out in three compartments namely, maternal serum (MS), colostrums and cord blood serum (CS) of healthy Indian mothers (n = 42) who delivered healthy normal neonates without any congenital anomalies at Bhabha Atomic Research Centre hospital, Mumbai. Fe, Zn, Cu in maternal serum, cord blood and colostrums were estimated by flame atomic absorption spectrometry while Se was determined by graphite furnace absorption spectrometry. It was seen that there was a significant difference in the level of trace elements in the three compartments. The average levels of Fe in the three compartments were 1,132 ± 519, 2,312 ± 789 and 1,183 ± 602 μg/L while Zn was 514 ± 149, 819 ± 224 and 7,148 ± 2,316 μg/L respectively. Mean Cu values were 1,614 ± 295, 301 ± 77 and 392 ± 174 μg/L respectively while Se values were 70 ± 15, 36 ± 10 and 23 ± 8 μg/L respectively. The results indicated a positive correlation of Fe and Zn concentrations in MS versus CS which were (r = 0.386), (r = 0.572) respectively and Fe levels in MS and colostrums (r = 0.235). A few inter element correlations were found within compartments. Zn and Se showed a negative correlation in both MS (r = −0.489) and colostrums (r = −0.258) while a positive inter correlation of Fe and Zn was seen in MS (r = 0.44) and in CS (r = 0.54). This study gave us an overview of the serum and colostrum values of mother and neonates in Indian population, data of which are scarce.     

Effect of sample type,centrifugation and storage conditions on vitamin D concentration

Introduction:

Studies about vitamin D [25(OH)D] stability in plasma are limited and preanalytical variables such as tube type may affect results. We aimed to evaluate effect of storage conditions, sample type and some preanalytical variables on vitamin D concentration.

Materials and methods:

Blood samples from 15 healthy subjects were centrifuged at different temperatures and stored under different conditions. Serum and plasma 25(OH)D difference, effect of centrifugation temperature and common storage conditions were investigated.

Results:

There was no difference between serum and plasma vitamin D concentration. Centrifugation temperature had no impact on vitamin D concentration. 25(OH)D is stable under common storage conditions: 4 hours at room temperature, 24 hours at 2–8 °C, 7 days at −20 °C, 3 months at −80 °C.

Conclusion:

Vitamin D does not require any special storage conditions and refrigeration. Both serum and plasma can be used for measurement.     

Hepatic Enzyme’s Reference Intervals and Their Modulating Factors in Western Indian Population

The reference intervals (RIs) of serum aminotransferases and Gamma-glutamyl transferase (GGT) have been established many years ago. Recent RIs are not available. The prospective study was conducted to re-evaluate the RIs of liver enzymes and the effect of demographic and anthropometric variables on them in western Indian population. A total of 1059 blood donors comprised the study population. Anthropometry and serum liver enzymes levels were measured. Subjects were categorized into normal weight and overweight by using body mass index (BMI) and waist circumference (WC). For RI determination, non-parametric methodology recommended by IFCC/CLSI was adopted. Mann–Whitney test and Spearman’s rank correlation were used for statistical analysis. Upper limit of normal reference value of liver enzymes were lower in female compared to male. (ALT—23.55 F vs 36.00 M, GGT—34.58 F vs 36.20 M) When RI of liver enzymes were calculated according to body mass index, the upper limit of normal of ALT and GGT were higher in overweight group compared to normal weight group. (ALT—38.00 vs 27.00 IU/L and GGT—37.59 vs 35.26 IU/L). In both male and female, liver enzymes correlated significantly with age. WC and BMI were positively correlated with AST, ALT and GGT in both subgroups and the correlation was stronger in male. Demographic factors should be considered for making liver enzyme tests more clinically relevant. Gender based partitioning should be adopted for serum alanine aminotransferase (ALT) and GGT reference values for Western Indian population.     

Increasing Glucose Concentrations Interfere with Estimation of Electrolytes by Indirect Ion Selective Electrode Method

The estimation of electrolytes like sodium (Na⁺), potassium (K⁺) and chloride (Cl⁻) using direct and indirect ion-selective electrodes (ISE) is a routine laboratory practice. Interferents like proteins, triglycerides, drugs etc. are known to affect the results. The present study was designed to look into the effect of increasing glucose concentrations on estimation of Na⁺, K⁺ and Cl⁻ by direct and indirect ISE. Pooled sera was mixed with glucose stock solution (20 g/dL) prepared in normal saline to obtain glucose concentrations ranging from ~100 to ~5000 mg/dL. Na⁺, K⁺ and Cl⁻ levels were estimated by direct and indirect ISE analyzers and results were statistically analysed using ANOVA and Pearson’s correlation. Similar experiment was also performed in 24 h urine sample from healthy subjects. Significant difference was observed between Na⁺ and Cl⁻ measurements by direct and indirect ISE, with indirect ISE values being consistently higher than direct ISE. Besides this, significant difference was observed amongst Na⁺ and Cl⁻ values from baseline values obtained by indirect ISE at glucose concentrations ≥2486 mg/dL. However, no such difference was observed with direct ISE. Na⁺ and Cl⁻ estimation by indirect ISE showed significant negative correlation with glucose concentration, more so, above ~2000 mg/dL. K⁺, however, showed no significant difference with varying glucose. Similar results were observed in 24 h urine samples with a significant difference observed amongst Na⁺ and Cl⁻ values at ≥2104 mg/dL glucose. Thus we conclude that high glucose concentrations interfere significantly in estimation of Na⁺ and Cl⁻ by indirect ISE in serum as well as urine.

Electronic supplementary material

The online version of this article (doi:10.1007/s12291-015-0522-0) contains supplementary material, which is available to authorized users.     

A case report of short-chain acyl-CoA dehydrogenase deficiency (SCADD)

Background

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare inherited mitochondrial fatty acid oxidation disorder associated with variations in the ACADS (Acyl-CoA dehydrogenase, C-2 to C-3 short chain) gene. SCADD has highly variable biochemical, genetic and clinical characteristics. Phenotypes vary from fatal metabolic decompensation to asymptomatic individuals.

Subject and methods

A Romani boy presented at 3 days after birth with hypoglycaemia, hypotonia and respiratory pauses with brief generalized seizures. Afterwards the failure to thrive and developmental delay were present. Organic acids analysis with gas chromatography-mass spectrometry (GS/MS) in urine and acylcarnitines analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in dried blood spot were measured. Deoxyribonucleic acid (DNA) was isolated from blood and polymerase chain reactions (PCRs) were performed for all exons. Sequence analysis of all exons and flanking intron sequences of ACADS gene was performed.

Results

Organic acids analysis revealed increased concentration of ethylmalonic acid. Acylcarnitines analysis showed increase of butyrylcarnitine, C4-carnitine. C4-carnitine was 3.5 times above the reference range (<0.68 µmol/L). Confirmation analysis for organic acids and acylcarnitine profile was performed on the second independent sample and showed the same pattern of increased metabolites. Sequence analysis revealed 3-bp deletion at position 310-312 in homozygous state (c.310_312delGAG). Mutation was previously described as pathogenic in heterozygous state, while it is in homozygous state in our patient.

Conclusions

In our case clinical features of a patient, biochemical parameters and genetic data were consistent and showed definitely SCAD deficiency.Key words: SCAD deficiency, short chain acyl-CoA dehydrogenase deficiency, screening, acylcarnitine, polymorphism, genetic     

Age-Related Changes in Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1) and Receptor 2 (sVEGFR2) in Healthy Japanese Subjects

We measured serum soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and receptor 2 (sVEGFR2) levels in healthy Japanese individuals in order to establish a reference value using a specific ELISA. Significant differences were observed in serum sVEGFR1 and sVEGFR2 levels between children and adults. To demonstrate the usefulness of the reference value for children, we measured serum sVEGFR1 and sVEGFR2 levels in children with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as a preliminary study. Serum sVEGFR2 levels in children with HUS were markedly higher than those in healthy children from the onset of D + HUS. The reference value for healthy children in the present study will allow normal and pathological conditions to be discriminated from each other in future study     

Prevalence of Vitamin D Deficiency in a Pediatric Hospital of Eastern India

Vitamin D deficiency is highly prevalent in Indian children of northern, western and southern states. Serum 25 hydroxy cholecalferol (ng/ml) was analyzed in 310 children and adolescents of pediatric hospital of Kolkata, India. Serum calcium (mg/dl), phosphorous (mg/dl) and alkaline phosphatase (IU/L) data was obtained. Median 25(OH)D was 19 ng/ml. 19.2 % of population had serum 25(OH)D < 10 ng/ml (severe deficiency), 52.9 % had <20 ng/ml (deficiency), 24.5 % had 20–29 ng/ml (insufficiency) and 22.6 % had >30 ng/ml (optimum). Deficiency was highest in adolescents (86.1 %), followed by school children (61.0 %), lowest in pre-school children (41.6 %). 25(OH)D concentrations was lowest in winters (P = 0.002) and spring (P = 0.03) compared to summer. There was no correlation with calcium (P = 0.99), phosphorous (P = 0.23) and ALP (P = 0.63). There is high prevalence of vitamin D deficiency in children and adolescents of eastern India. Prevalence was lower in younger subjects. 25(OH)D did not correlate with bone mineral markers.