Alcohol consumption and serum metabolite concentrations in young women.

PURPOSE: Alcohol consumption is an established breast cancer risk factor, though further research is needed to advance our understanding of the mechanism underlying the association. We used global metabolomics profiling to identify serum metabolites and metabolic pathways that could potentially mediate the alcohol-breast cancer association. METHODS: A cross-sectional analysis of reported alcohol consumption and serum metabolite concentrations was conducted among 211 healthy women 25-29 years old who participated in the Dietary Intervention Study in Children 2006 Follow-Up Study (DISC06). Alcohol-metabolite associations were evaluated using multivariable linear mixed-effects regression. RESULTS: Alcohol was significantly (FDR p < 0.05) associated with several serum metabolites after adjustment for diet composition and other potential confounders. The amino acid sarcosine, the omega-3 fatty acid eicosapentaenoate, and the steroid 4-androsten-3beta,17beta-diol monosulfate were positively associated with alcohol intake, while the gamma-tocopherol metabolite gamma-carboxyethyl hydroxychroman (CEHC) was inversely associated. Positive associations of alcohol with 2-methylcitrate and 4-androsten-3beta,17beta-diol disulfate were borderline significant (FDR p < 0.10). Metabolite set enrichment analysis identified steroids and the glycine pathway as having more members associated with alcohol consumption than expected by chance. CONCLUSIONS: Most of the metabolites associated with alcohol in the current analysis participate in pathways hypothesized to mediate the alcohol-breast cancer association including hormonal, one-carbon metabolism, and oxidative stress pathways, but they could also affect risk via alternative pathways. Independent replication of alcohol-metabolite associations and prospective evaluation of confirmed associations with breast cancer risk are needed.

Plasma methylcitric acid and its correlations with other disease biomarkers: The impact in the follow up of patients with propionic and methylmalonic acidemia.

Methylcitric acid (MCA) analysis has been mainly utilized for the diagnosis of propionate disorders or as a second-tier test in newborn screening, but its utility for patients monitoring still needs to be established. We explored the potential contribution of MCA in the long-term management of organic acidurias. We prospectively evaluated plasma MCA and its relationship with disease biomarkers, clinical status, and disease burden in 22 patients, 13 with propionic acidemia (PA) and nine with methylmalonic acidemia (MMA) on standard treatment and/or after transplantation. Samples were collected at scheduled routine controls or during episodes of metabolic decompensation (MD), 10 patients were evaluated after transplantation (six liver, two combined liver and kidney, 2 kidney). MCA levels were higher in PA compared to MMA and its levels were not influenced by the clinical status (MD vs well state). In MMA, MCA was higher in elder patients and, along with fibroblast growth factor 21 (FGF21) and plasma methylmalonic acid, negatively correlated with GFR. In both diseases, MCA correlated with ammonia, glycine, lysine, C3, and the C3/C2, C3/C16 ratios. The disease burden showed a direct correlation with MCA and FGF21, for both diseases. All transplanted patients showed a significant reduction of MCA in comparison to baseline values, with some differences dependent on the type of transplantation. Our study provided new insights in understanding the disease pathophysiology, showing similarities between MCA and FGF21 in predicting disease burden, long-term complications and in evaluating the impact of organ transplantation.

Measurement, model prediction and uncertainty quantification of plasma clearance of cerium citrate in humans.

Double tracer studies in healthy human volunteers with stable isotopes of cerium citrate were performed with the aim of investigating the gastro-intestinal absorption of cerium (Ce), its plasma clearance and urinary excretion. In the present work, results of the clearance of Ce in blood plasma are shown after simultaneous intravenous and oral administration of a Ce tracer. Inductively coupled plasma mass spectrometry was used to determine the tracer concentrations in plasma. The results show that about 80% of the injected Ce citrate cleared from the plasma within the 5 mins post-administration. The data obtained are compared to a revised biokinetic model of Ce, which was initially developed by the International Commission on Radiological Protection (ICRP). The measured plasma clearance of Ce citrate was mostly consistent with that predicted by the ICRP biokinetic model. Furthermore, in an effort to quantify the uncertainty of the model prediction, the laboratory animal data on which the ICRP biokinetic Ce model is based, was analyzed. The measured plasma clearance and its uncertainty was also compared to the plasma clearance uncertainty predicted by the model. It was found that the measured plasma clearance during the first 15 min after administration is in a good agreement with the modelled plasma clearance. In general, the measured clearance falls inside the 95% confidence interval predicted by the biokinetic model.

Rapid and cost-effective LC-MS/MS method for determination of hydroxycitric acid in plasma: Application in the determination of pharmacokinetics in commercial Garcinia preparations.

Garcinia cambogia is one of the most commonly used anti-obesity dietary supplements, and hydroxycitric acid (HCA) is a major constituent in the commercial preparations of Garcinia. High doses of HCA are often consumed without much awareness of its pharmacokinetic and toxicokinetic parameters, and therefore, a complete understanding of its effects is lacking. The first step in understanding these parameters is the availability of a reliable bioanalytical method. Here, we present the first report on a UPLC-MS/MS method for analysis of HCA in rat plasma after a simplified and cost-effective protein precipitation. Chromatographic separation of the analytes in the supernatant was achieved using hydrophilic interaction liquid chromatography, where mass parameters were optimized and a rapid 5-min quantitative assay was developed. The method was highly sensitive, accurate, precise and linear in the concentration range of 10.5-5000 ng/mL (validated according to the United States Food and Drug Administration guidelines). Further, the method was successfully used to describe the pharmacokinetic profile of HCA in rat plasma after the administration of pure HCA and commercial Garcinia preparations.

Role of Functional Biomarkers to Identify Early Vitamin B12 Deficiency in Patients with Sleeve Gastrectomy: A Cross-Sectional Study.

Background and objectives: Although laparoscopic sleeve gastrectomy (LSG) is effective for obesity management, postoperative vitamin B12 (B12) deficiency is of major concern. In this cross-sectional study, we assessed the levels of B12 and its related functional biomarkers, namely, total homocysteine (tHcy), methylmalonic acid (MMA), folate, methylcitric acid (MCA), and hemoglobin (Hb), in one-year postoperative LSG patients and matched controls. Materials and Methods: Plasma B12, tHcy, MMA, folate, and MCA were measured in matched controls (n = 66) and patients (n = 71) using validated liquid chromatography-tandem mass spectrometry techniques and protocols in the United Arab Emirates (UAE). Results: The median B12 concentration in patients (177 pmol/L) was significantly lower (p < 0.001) than in the controls (334.7 pmol/L). The tHcy and MMA levels were significantly increased (p < 0.001 and p = 0.011, respectively) and folate levels were significantly decreased (p = 0.001) in the LSG patients compared to the controls. Interestingly, no significant difference in MCA levels were observed between the two groups. The levels of tHcy and MMA were concomitantly increased with the decreased folate levels in postoperative LSG patients when compared with the controls. The Hb levels were significantly lower in males and females in the patient group compared with those in the control group, respectively (p = 0.005 and p = 0.043). Conclusions: This is the first report of serum levels of B12 and its functional biomarkers in postoperative LSG patients among a local population from the UAE. Our findings revealed significant alterations of the B12 biomarkers, total B12, MMA, and tHcy in one-year postoperative LSG patients.

An Optimized and Sensitive Pharmacokinetic Quantitative Method of Investigating Gastrodin, Parishin, and Parishin B, C and E in Beagle Dog Plasma using LC-MS/MS after Intragastric Administration of Tall Gastrodia Capsules.

Gastrodia elata Blume, called Tianma in China, has been widely used to treat headaches, convulsions and epilepsy for thousands of years. In the present study, a series of optimizations were employed to develop a rapid, sensitive, and reliable high-performance liquid chromatography-triple quadrupole mass spectrometry method, which was then used for the simultaneous determination of gastrodin, parishin, parishin B, parishin C and parishin E in beagle dog plasma after intragastric administration of tall Gastrodia capsules (Tianma brand). The chromatographic separation was achieved on a C18 column with gradient elution by using a mixture of 0.4% formic acid aqueous solution and acetonitrile as the mobile phase at a flow rate of 0.15 mL/min. A tandem mass spectrometric detection was conducted using multiple-reaction monitoring (MRM) via electrospray ionization (ESI) source in negative ionization mode. Samples were pre-treated by a single-step protein precipitation with methanol, and bergenin was used as internal standard (IS). Under the optimized conditions, the lower limit of quantification (LLOQ) was 0.10 ng/mL for gastrodin, 0.40 ng/mL for parishin B, 0.02 ng/mL for parishin E and 0.20 ng/mL for parishin and parishin C, all of which previously were the highest levels of sensitivity. The methods were optimized for selectivity, calibration curves, accuracy and precision. Extraction recoveries, matrix effects and stability were within acceptable ranges. Pharmacokinetic parameters of the tested substances were also quantitatively determined. Finally, a possible metabolic pathway was induced based on correlations obtained from quantitative and qualitative data analysis in vivo.

Distribution of citrate and citrate infusion rate during donor plasmaphereses.

BACKGROUND: The purpose of this study was to describe the distribution of trisodium-citrate 4% (TSC) anticoagulant (AC) between the product and the donors undergoing plasma donation. SUBJECTS AND METHODS: Data of 32 regular donors of plasma initially collected for a study published in 2010 were re-analyzed to determine the amount of citrate received by the donor and the citrate infusion rate (CIR) in mg/kg/min to the donor. Donor plasmaphereses (DP) were performed with the automated Haemonetics plasma collecting system 2 (PCS2). Plasma volume was programmed at 760 ml including AC. CIR was calculated from citrate received by the donors divided by the body weight over time. RESULTS: 130 ± 12 ml TSC was used for 760 ml plasma. An average of 110 ml TSC or 84.6% of citrate load was in collected plasma and not given to the donor. From the difference of 20 ml or 514 mg citrate an average CIR of 0.16 mg/kg/min was calculated. CONCLUSION: The total amount of citrate received by the donor is minimal and the average CIR is below the critical level of 1 mg/kg/min.

Induced Alkalosis and Gastrointestinal Symptoms After Sodium Citrate Ingestion: a Dose-Response Investigation.

Sodium citrate induces alkalosis and can provide a performance benefit in high-intensity exercise. Previous investigations have been inconsistent in the ingestion protocols used, in particular the dose and timing of ingestion before the onset of exercise. The primary aim of the current study was to quantify blood pH, blood bicarbonate concentration and gastrointestinal symptoms after ingestion of three doses of sodium citrate (500 mg⋅kg-1, 700 mg⋅kg-1 and 900 mg⋅kg-1). Thirteen participants completed four experimental sessions, each consisting of a different dose of sodium citrate or a taste-matched placebo solution. Blood pH and blood bicarbonate concentration were measured at 30-min intervals via analysis of capillary blood samples. Gastrointestinal symptoms were also monitored at 30-min intervals. Statistical significance was accepted at a level of p < .05. Both measures of alkalosis were significantly greater after ingestion of sodium citrate compared with placebo (p < .001). No significant differences in alkalosis were found between the three sodium citrate doses (p > .05). Peak alkalosis following sodium citrate ingestion ranged from 180 to 212 min after ingestion. Gastrointestinal symptoms were significantly higher after sodium citrate ingestion compared with placebo (p < .001), while the 900 mg.kg-1 dose elicited significantly greater gastrointestinal distress than 500 mg⋅kg-1 (p = .004). It is recommended that a dose of 500 mg⋅kg-1 of sodium citrate should be ingested at least 3 hr before exercise, to achieve peak alkalosis and to minimize gastrointestinal symptoms before and during exercise.

Effect of organo and inorganic lithium salt on human blood plasma glutathione- A comparative study.

Investigation of toxicological effect of various metals is the field of interest for toxicological scientists since four to five decades and especially the toxicological effect of those drugs containing metals and there use is common because there is no other choice except to use these metal containing drugs. Inorganic as well as organic salts of lithium are commonly used in prophylaxis and treatments of many psychiatric disorders. The aim of the present study was to see the difference between the effect of organic and inorganic salt of lithium commonly used in psychiatric disorders on the GSH of human blood plasma. It is the scientific fact that ionic dissociation of organic and inorganic salts of any metal is always quite different hence to prove this fact, the effect of lithium citrate (organic salt of lithium) and lithium carbonate (inorganic salt of lithium) was investigated on human blood plasma GSH to find the difference between the effect of two. Ellman's method was used for the quantification of glutathione contents in plasma. It was found that lithium citrate decrease plasma GSH contents less than lithium carbonate indicating that organic salts of lithium are safe than inorganic salts of lithium when are used in psychiatric disorders. Further to analyze the effect of organic and inorganic salt of lithium on blood plasma GSH with the increase in incubation time was also evaluated and was found that both concentration and time dependent effect of organic salt of lithium shows that this salt has decreased plasma GSH contents of human blood less than inorganic salt of lithium either by promoting oxidation of GSH into GSSG or by lithium glutathione complex formation. These results suggest the physicians that the use of organic lithium salts is much safer than inorganic salts of lithium in terms of depletion of blood plasma GSH contents.

Pharmacokinetic study of Gastrodia elata in rats.

The pharmacokinetics of parishin, gastrodin, Gastrodia elata extract and Rhizoma Gastrodiae capsule was investigated by intragastric and/or intravenous administration to rats. Parishin was metabolized into nine metabolites after intravenous administration, and the area under the curve (AUC0-∞) of parishin and its metabolites (except parishin G and parishin E) increased nonlinearly from 72.5 to 220 mg/kg. When combining regression equation with the AUC0-∞ and dose of gastrodin injection, the percent conversion of parishin to gastrodin was obtained as 50 %. Based on multi-active metabolites of parishin in vivo, integrated pharmacokinetic mode was established. It is notable that each metabolite from parishin shares the similar metabolic process at three dosages of parishin and the bioavailability of parishin was approximately 14 %. The integrated pharmacokinetic mode was successfully applied to evaluate the holistic pharmacokinetics of gastrodin injection, G. elata extract and Rhizoma Gastrodiae capsule. The results showed that the holistic pharmacokinetics of gastrodin injection and G. elata extract was closed to that of gastrodin, but for parishin and Rhizoma Gastrodiae capsule, integrated pharmacokinetic parameters were more suitable to evaluate its holistic pharmacokinetics. Graphical abstract Pharmacokinetic study of Gastrodia elata in rats.

Analysis of the metabolic profile of parishin by ultra-performance liquid chromatography/quadrupole-time of flight mass spectrometry.

Parishin is a dominant active ingredient originating from Gastrodia elata Blume, and has good neuroprotective effects against brain disorders. In the present study, the metabolic profile of parishin by in vitro and in vivo experiments was investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC/Q-TOF MS) combined with an automated MS(E) technique. By comparison with reference compounds, accurate mass measurement, the characteristic fragmentation patterns of the parent drug parishin and gastrodin and relevant bio-transformation knowledge, 14 metabolites (seven hydrolyzates and seven derivatives of gastrodin) were detected and identified in rat plasma and urine after intragastric administration of parishin, including processes of hydrolyzation, oxidation, sulfation and glucuronidation. According to the proposed metabolic pathways of parishin, in vitro hydrolytic experiments and metabolic study of gastrodin in rat plasma, it can be inferred that parishin mainly functions as a prodrug and undergoes hydrolysis before being absorbed into the blood. The hydrolyzate, mainly gastrodin, was involved in further metabolism, which was responsible for pharmacological activities of parishin. In conclusion, this work provides valuable information on parishin metabolism using a rapid and reliable UHPLC/Q-TOF MS method, which could be widely used for the metabolic investigation of natural product.

Clinical evaluation of a model for prediction of end-dialysis systemic ionized calcium concentration in citrate hemodialysis.

BACKGROUND/AIM: Citrate anticoagulation in hemodialysis (HD) is increasingly drawing attention in the nephrology community. One of the major deterrents to a more widespread use are the monitoring requirements for fear of systemic calcium derangements. Means of accurately predicting systemic ionized calcium (iCa) may help to overcome this challenge. We have previously presented a mathematical model of regional citrate anticoagulation (RCA) to address this need. Here, we present a refined model and show results in an independent validation cohort of maintenance HD patients on Citrasate®, a calcium- and citrate-containing dialysate. METHODS: A hybrid RCA model was developed, comprising the previously published 'native' RCA model and a statistical correction based on levels of alkaline phosphatase as a marker of bone turnover. The model was validated in 120 patients on Citrasate, a dialysate containing 0.8 mmol/l citrate and 1.125 mmol/l calcium. Systemic iCa was measured at the beginning and end of one HD treatment in each subject. Serum iCa predictions were compared between our previously published model and the new hybrid model. RESULTS: On average, the hybrid model predicted end-HD systemic iCa with an error (predicted - measured) of 0.028 mmol/l, compared to -0.051 mmol/l with the previously published model. There were only 4 subjects out of the 120 analyzed in whom the prediction error was <-0.1 mmol/l, and only 6 in whom the error was >+0.1 mmol/l (max: +0.13 mmol/l). CONCLUSION: This study demonstrates that the novel hybrid model is an improvement over the previously published model and that it is capable of predicting end-dialysis systemic iCa levels with improved accuracy and precision even in a citrate dialysis setting which was much different from the original derivation cohort.

Vision changes after spaceflight are related to alterations in folate- and vitamin B-12-dependent one-carbon metabolism.

Approximately 20% (7 of 38) of astronauts on International Space Station (ISS) missions have developed measurable ophthalmic changes after flight. This study was conducted to determine if the folate- and vitamin B-12-dependent 1-carbon metabolic pathway is altered in these individuals. Since 2006, we have conducted experiments on the ISS to evaluate nutritional status and related biochemical indices of astronauts before, during, and after flight. Data were modeled to evaluate differences between individuals with ophthalmic changes (n = 5) and those without them (n = 15), all of whom were on ISS missions of 48-215 d. We also determined whether mean preflight serum concentrations of the 1-carbon metabolites and changes in measured cycloplegic refraction after flight were associated. Serum homocysteine (Hcy), cystathionine, 2-methylcitric acid (2MCA), and methylmalonic acid concentrations were 25-45% higher (P < 0.001) in astronauts with ophthalmic changes than in those without them. These differences existed before, during, and after flight. Preflight serum concentrations of Hcy and cystathionine, and mean in-flight serum folate, were correlated with change (postflight relative to preflight) values in refraction (P < 0.05), and preflight serum concentrations of 2MCA tended to be associated (P = 0.06) with ophthalmic changes. The biochemical differences observed in crewmembers with vision issues strongly suggest that their folate- and vitamin B-12-dependent 1-carbon transfer metabolism was affected before and during flight. The consistent differences in markers of 1-carbon metabolism between those who did and those who did not develop changes in vision suggest that polymorphisms in enzymes of this pathway may interact with microgravity to cause these pathophysiologic changes.

Precise pH measuring of platelet concentrates containing additive solution--the impact of the temperature.

BACKGROUND AND OBJECTIVES: Blood gas analysers measuring pH at 37°C (pH37) are widely used for pH determination of platelet (PLT) concentrates (PCs). For reporting pH at 22°C (pH22), converting of pH37 using the correct conversion factor is mandatory. For PCs stored in PLT additive solution (PAS), such conversion factors are not yet widely available. We studied pH in samples of PCs with different PAS/plasma ratios during warming from 22 to 37°C. MATERIALS AND METHODS: We measured pH in 39 samples containing modified PAS-III (PAS-IIIM) with a plasma carryover of 20%, 30% or 40% or no PAS-IIIM. Differences between pH22 and pH37 (dpH) were compared within and between study groups. Correlation between pH22 and dpH was tested. Additional measurements in 33 samples with three different PLT counts were performed to study the influence of PLT count on dpH. RESULTS: pH22 and pH37 within each group and dpH or dpH/dT between study groups differed significantly. The dpH was 0·135 ± 0·040, 0·021 ± 0·009, 0·033 ± 0·011 and 0·048 ± 0·017 for samples containing 100%, 20%, 30% or 40% plasma, respectively. Correlation between dpH and pH22 was strong in 100% (r = 0·696, P < 0·001), weaker in 30% and 40% (r = 0·367, P = 0·022 and r = 0·345, P = 0·032, respectively) and not existing in 20% plasma (r = 0·153, P = 0·354). PLT count did not influence the dpH significantly. CONCLUSION: The dpH is dependent on different PAS-IIIM/plasma ratios and pH range. For precise reporting of pH22, the respective dpH must be used if converting is necessary. Preferably, the pH should be reported at 37°C or measured directly at 22°C.

A novel method for measuring catheter lock spillage: an in vitro study.

Catheters are widely used for blood purification, parenteral nutrition, and for the infusion of drugs. Previous work on catheter lock spillage has focused on the theory and in vitro demonstration of catheter lock spillage caused by the laminar flow profile and by fluid exchange caused by density differences. This work describes an in vitro test with a method that potentially allows measurement of catheter lock spillage in vivo without sampling. The method is based on the change of the electrical resistance of the catheter when the lock solution is injected. This method was tested in vitro with human blood at 36°C using 46.7% trisodium citrate as catheter lock solution. The catheter tip was placed in a beaker filled with whole blood. A stainless steel rod in the beaker served as one electrode and an Arrow-Johans ECG adapter, which was placed on the distal end of the catheter, served as a second electrode. Conductivity was measured with a 5V (rms) 310 Hz sinus voltage and a 10 kOhm resistor in series to the catheter. The driving voltage and the voltage drop at the catheter was continuously measured with a program written under LabView (National Instruments), and the results were converted into mean trisodium citrate concentrations. Within 20 min, the mean trisodium citrate concentration in the catheter decreased to less than 5%. Unlike the previous methods used for catheter lock spillage measurement, this principle can be employed to measure the time course of catheter lock spillage in vivo.

Metabolite Biomarkers of CKD Progression in Children.

BACKGROUND AND OBJECTIVES: Metabolomics facilitates the discovery of biomarkers and potential therapeutic targets for CKD progression. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We evaluated an untargeted metabolomics quantification of stored plasma samples from 645 Chronic Kidney Disease in Children (CKiD) participants. Metabolites were standardized and logarithmically transformed. Cox proportional hazards regression examined the association between 825 nondrug metabolites and progression to the composite outcome of KRT or 50% reduction of eGFR, adjusting for age, sex, race, body mass index, hypertension, glomerular versus nonglomerular diagnosis, proteinuria, and baseline eGFR. Stratified analyses were performed within subgroups of glomerular/nonglomerular diagnosis and baseline eGFR. RESULTS: Baseline characteristics were 391 (61%) male; median age 12 years; median eGFR 54 ml/min per 1.73 m2; 448 (69%) nonglomerular diagnosis. Over a median follow-up of 4.8 years, 209 (32%) participants developed the composite outcome. Unique association signals were identified in subgroups of baseline eGFR. Among participants with baseline eGFR ≥60 ml/min per 1.73 m2, two-fold higher levels of seven metabolites were significantly associated with higher hazards of KRT/halving of eGFR events: three involved in purine and pyrimidine metabolism (N6-carbamoylthreonyladenosine, hazard ratio, 16; 95% confidence interval, 4 to 60; 5,6-dihydrouridine, hazard ratio, 17; 95% confidence interval, 5 to 55; pseudouridine, hazard ratio, 39; 95% confidence interval, 8 to 200); two amino acids, C-glycosyltryptophan, hazard ratio, 24; 95% confidence interval 6 to 95 and lanthionine, hazard ratio, 3; 95% confidence interval, 2 to 5; the tricarboxylic acid cycle intermediate 2-methylcitrate/homocitrate, hazard ratio, 4; 95% confidence interval, 2 to 7; and gulonate, hazard ratio, 10; 95% confidence interval, 3 to 29. Among those with baseline eGFR <60 ml/min per 1.73 m2, a higher level of tetrahydrocortisol sulfate was associated with lower risk of progression (hazard ratio, 0.8; 95% confidence interval, 0.7 to 0.9). CONCLUSIONS: Untargeted plasma metabolomic profiling facilitated discovery of novel metabolite associations with CKD progression in children that were independent of established clinical predictors and highlight the role of select biologic pathways.

Metformin, valproic acid, and starvation induce seizures in a patient with partial SLC13A5 deficiency: a case of pharmaco-synergistic heterozygosity.

SLC13A5/NaCT is a sodium-coupled citrate transporter expressed in the plasma membrane of the liver, testis, and brain. In these tissues, SLC13A5 has important functions in the synthesis of fatty acids, cholesterol, and neurotransmitters. In recent years, patients homozygous for recessive mutations in SLC13A5, known as SLC13A5 deficiency [early infantile epileptic encephalopathy-25 (EIEE-25)], exhibit severe global developmental delay, early-onset intractable seizures, spasticity, and amelogenesis imperfecta affecting tooth development. Although the pathogenesis of SLC13A5 deficiency remains not clearly understood, cytoplasmic citrate deficits, decreased energy status in neurons, and citrate-zinc chelation are hypothesized to explain the neurological deficits. However, no study has examined the possibility of specific pharmacological drugs and/or lifestyle changes synergizing with heterozygosity of SLC13A5 deficiency to increase the risk of EIEE-25 clinical phenotype. Here, we report on a heterozygous SLC13A5-deficient patient who demonstrated evidence of pharmaco-synergistic heterozygosity upon administration of metformin, valproic acid, and starvation. The report illustrates the importance of careful consideration of the potential adverse effects of specific pharmacological treatments in patients with heterozygosity for disease-causing recessive mutations in SLC13A5.

Determination of total homocysteine, methylmalonic acid, and 2-methylcitric acid in dried blood spots by tandem mass spectrometry.

BACKGROUND: Newborn screening (NBS) for inborn errors of propionate, methionine, and cobalamin metabolism relies on finding abnormal concentrations of methionine and propionylcarnitine. These analytes are not specific for these conditions and lead to frequent false-positive results. More specific markers are total homocysteine (tHCY), methylmalonic acid (MMA), and methylcitric acid (MCA), but these markers are not detected by current NBS methods. To improve this situation, we developed a method for the detection of tHCY, MMA, and MCA in dried blood spots (DBSs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The analytes were extracted from a single 4.8-mm DBS punch with acetonitrile:water:formic acid (59:41:0.42) containing dithiothreitol and isotopically labeled standards (d(3)-MMA, d(3)-MCA, d(8)-homocystine). The extract was dried and treated with 3 N HCl in n-butanol to form butylesters. After evaporation of the butanol, the residue was reconstituted and centrifuged and the supernatant was subjected to LC-MS/MS analysis. Algorithms were developed to apply this method as an efficient and effective second-tier assay on samples with abnormal results by primary screening. RESULTS: The 99th percentiles determined from the analysis of 200 control DBSs for MMA, MCA, and HCY were 1.5, 0.5, and 9.8 µmol/L, respectively. Since 2005, prospective application of this second-tier analysis to 2.3% of all NBS samples led to the identification of 13 affected infants. CONCLUSIONS: Application of this assay reduced the false-positive rate and improved the positive predictive value of NBS for conditions associated with abnormal propionylcarnitine and methionine concentrations.

Analytical recovery of folate degradation products formed in human serum and plasma at room temperature.

Folate is not stable in serum and plasma. This may impair laboratory diagnostics and distort the outcome of epidemiological studies on folate and chronic diseases. The present study was designed to determine the kinetics of folate loss in human serum and plasma (collected into tubes containing EDTA, heparin, or citrate) at room temperature and the recovery of folate as 4-alpha-hydroxy-5-methyltetrahydrofolate (hmTHF) or p-aminobenzoylglutamate (pABG) equivalents. Different folate species and pABG were determined by liquid chromatography-tandem MS and microbiologically active folate was measured by a Lactobacillus rhamnosus assay. Concentrations of 5mTHF and microbiologically active folate had a parallel and rapid decrease in EDTA plasma to approximately 60% of the initial concentration after 24 h. In serum, heparin plasma, and citrate plasma, folate decreased more slowly to approximately 50% after 192 h. The loss of 5mTHF that occurred within 48 h was totally recovered as hmTHF. Folate measured as pABG equivalents decreased slowly to approximately 80% in 192 h and the decline was essentially matrix independent. In conclusion, the degradation of 5mTHF and microbiologically active folate in serum and plasma at room temperature can largely be corrected for by determining hmTHF or measuring folate as pABG equivalents. Moreover, results obtained using conventional folate assays may be biased by improper sample handling or if samples contained high concentrations of hmTHF.