Elaidate, an 18-carbon trans-monoenoic fatty acid, inhibits ß-oxidation in human peripheral blood macrophages.

Consumption of trans-unsaturated fatty acids promotes atherosclerosis, but whether degradation of fats in macrophages is altered by trans-unsaturated fatty acids is unknown. We compared the metabolism of oleate (C18:1Δ9-10 cis; (Z)-octadec-9-enoate), elaidate (C18:Δ9-10 trans; (E)-octadec-9-enoate), and stearate (C18:0, octadecanoate) in adherent peripheral human macrophages. Metabolism was followed by measurement of acylcarnitines in cell supernatants by MS/MS, determination of cellular fatty acid content by GC/MS, and assessment of ß-oxidation rates using radiolabeled fatty acids. Cells incubated for 44 h in 100 µM elaidate accumulated more unsaturated fatty acids, including both longer- and shorter-chain, and had reduced C18:0 relative to those incubated with oleate or stearate. Both C12:1 and C18:1 acylcarnitines accumulated in supernatants of macrophages exposed to trans fats. These results suggested ß-oxidation inhibition one reaction proximal to the trans bond. Comparison of [1-(14)C]oleate to [1-(14)C]elaidate catabolism showed that elaidate completed the first round of fatty acid ß-oxidation at rates comparable to oleate. Yet, in competitive ß-oxidation assays with [9,10-(3)H]oleate, tritium release rate decreased when unlabeled oleate was replaced by the same quantity of elaidate. These data show specific inhibition of monoenoic fat catabolism by elaidate that is not shared by other atherogenic fats.

Quantitation of phenylalanine and tyrosine from dried Blood/Plasma spots with impregnated stable isotope internal standards (SIIS) by FIA-SRM.

BACKGROUND: Dried Blood Spot (DBS) analysis has been used for identification and quantification of diseases and disorders in large populations. Simply collecting blood or plasma samples on cotton paper, followed with an organic solvent extraction, many small molecules can be detected and quantified. In a typical procedure of DBS analysis in newborn screening, stable isotope internal standards (SIIS) are added to extraction solvent as a reference. However, this way of employing SIIS does not reflect extraction efficiency, or protein binding issues, nor does it reflect potential degradation that could occur. In addition, punched-out discs from larger DBS are known to have imprecision typically ≥ 15%. METHODS: We developed and tested an approach, internal quantitative DBS (iqDBS), which delivers an exact volume of whole blood or plasma to a paper disc that is impregnated with a dried concentration of SIIS for quantitation. Amino acids were derivatized to make butyl esters and measured using Flow Injection Analysis with Selected Reaction Monitoring (FIA-SRM). RESULTS: We demonstrated with phenylalanine and tyrosine improved sensitivity and accuracy by applying iqDBS. CONCLUSIONS: We established a new method for quantitative analysis of small molecules from dried blood spots that incorporates stable isotope internal standard at the time of blood collection.

Evaluation of asymmetric dimethylarginine, arginine, and carnitine metabolism in pediatric sepsis.

OBJECTIVE: Increased plasma concentrations of the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine, decreased arginine bioavailability, and mitochondrial dysfunction have been reported in adult sepsis. We studied whether asymmetric dimethylarginine, arginine, and carnitine metabolism (a measure of mitochondrial dysfunction) are altered in pediatric sepsis and whether these are clinically useful biomarkers. DESIGN: : Prospective, observational study. SETTING: Pediatric intensive care unit at an academic medical center. PATIENTS: : Ninety patients ≤ 18 yrs old, 30 with severe sepsis or septic shock, compared with 30 age-matched febrile and 30 age-matched healthy control subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma asymmetric dimethylarginine and whole blood arginine, citrulline, ornithine, and acylcarnitine:free carnitine ratio were measured daily for septic patients and once for control subjects using tandem mass spectrometry. Plasma asymmetric dimethylarginine concentration (median; interquartile range µmol/L) on day 1 was lower in severe sepsis and septic shock (0.38; 0.30-0.56) compared with febrile (0.45; 0.40-0.59) and healthy (0.60; 0.54-0.67) control subjects (p < .001), although decreased asymmetric dimethylarginine was predominantly found in neutropenic patients. Day 1 arginine was lower in septic (10; interquartile range, 7-20 µmol/L) compared with healthy patients (32; interquartile range, 23-40; p < .001), and the arginine:ornithine ratio was decreased in sepsis, indicating increased arginase activity (an alternative pathway for arginine metabolism). The arginine:asymmetric dimethylarginine and acylcarnitine:free carnitine ratios did not differ between septic and control patients. Asymmetric dimethylarginine was inversely correlated with organ dysfunction by Pediatric Logistic Organ Dysfunction score (r = -0.50, p = .009), interleukin-6 (r = -0.55, p = .01), and interleukin-8 (r = -0.52, p = .03) on admission. Arginine, arginine:asymmetric dimethylarginine, and acylcarnitine:free carnitine were not associated with organ dysfunction or outcomes. CONCLUSIONS: Asymmetric dimethylarginine was decreased in pediatric sepsis and was inversely associated with inflammation and organ dysfunction. This suggests that inhibition of nitric oxide synthase by asymmetric dimethylarginine accumulation is unlikely to impact sepsis pathophysiology in septic children despite decreased arginine bioavailability. We did not find an association of asymmetric dimethylarginine with altered carnitine metabolism nor were asymmetric dimethylarginine, arginine, and acylcarnitine:free carnitine useful as clinical biomarkers.

Progressive Metabolic Abnormalities Associated with the Development of Neonatal Bronchopulmonary Dysplasia.

Objective: To assess the longitudinal metabolic patterns during the evolution of bronchopulmonary dysplasia (BPD) development. Methods: A case-control dataset of preterm infants (<32-week gestation) was obtained from a multicenter database, including 355 BPD cases and 395 controls. A total of 72 amino acid (AA) and acylcarnitine (AC) variables, along with infants' calorie intake and growth outcomes, were measured on day of life 1, 7, 28, and 42. Logistic regression, clustering methods, and random forest statistical modeling were utilized to identify metabolic variables significantly associated with BPD development and to investigate their longitudinal patterns that are associated with BPD development. Results: A panel of 27 metabolic variables were observed to be longitudinally associated with BPD development. The involved metabolites increased from 1 predominant different AC by day 7 to 19 associated AA and AC compounds by day 28 and 16 metabolic features by day 42. Citrulline, alanine, glutamate, tyrosine, propionylcarnitine, free carnitine, acetylcarnitine, hydroxybutyrylcarnitine, and most median-chain ACs (C5:C10) were the most associated metabolites down-regulated in BPD babies over the early days of life, whereas phenylalanine, methionine, and hydroxypalmitoylcarnitine were observed to be up-regulated in BPD babies. Most calorie intake and growth outcomes revealed similar longitudinal patterns between BPD cases and controls over the first 6 weeks of life, after gestational adjustment. When combining with birth weight, the derived metabolic-based discriminative model observed some differences between those with and without BPD development, with c-statistics of 0.869 and 0.841 at day 7 and 28 of life on the test data. Conclusions: The metabolic panel we describe identified some metabolic differences in the blood associated with BPD pathogenesis. Further work is needed to determine whether these compounds could facilitate the monitoring and/or investigation of early-life metabolic status in the lung and other tissues for the prevention and management of BPD.

Impact of vaccination during pregnancy and staphylococci concentration on the presence of Bacillus cereus in raw human milk.

OBJECTIVE: This study aimed to determine whether vaccination during pregnancy, prematurity, and staphylococci concentration influenced the presence of B. cereus or staphylococcal enterotoxins (SEs) in raw human milk from healthy mothers. STUDY DESIGN: Human milk samples were collected from 152 healthy women. B. cereus, S. aureus and coagulase-negative staphylococci (CNS) were enumerated using selective agar culture media. The detection of B. cereus spores and SEs were determined using ELISA. RESULTS: CNS and B. cereus concentrations in milk from non-vaccinated mothers were higher than that from mothers vaccinated during pregnancy, but S. aureus did not differ. Prematurity did not affect B. cereus or staphylococci in human milk. S. aureus and CNS concentrations in human milk with the presence of B. cereus were higher than that with the absence of B. cereus. Viable B. cereus was present in 9.2% of raw human milk samples whereas SEs were not detected in any samples. CONCLUSIONS: Vaccination during pregnancy and low concentration of staphylococci could reduce the risk of B. cereus in raw human milk. The screening of B. cereus in raw human milk must be performed before pasteurization to reduce the risk of B. cereus infection in preterm infants.

Mass spectrometry in newborn and metabolic screening: historical perspective and future directions.

The growth of mass spectrometry (MS) in clinical chemistry has primarily occurred in two areas: the traditional clinical chemistry areas of toxicology and therapeutic drug monitoring and more recent, human genetics and metabolism, specifically inherited disorders of intermediary metabolism and newborn screening. Capillary gas chromatography and electrospray tandem MS are the two most common applications used to detect metabolic disease in screening, diagnostics and disease monitoring of treated patients. A few drops of blood from several million newborn infants are screened annually throughout the world making this the largest application of MS in medicine. Understanding the technique, how it grew from a few dozen samples per week in the early 1990s to increasing daily volume today will provide important information for new tests that either expand newborn screening or screening in other areas of metabolism and endocrinology. There are numerous challenges to the further expansion of MS in clinical chemistry but also many new opportunities in closely related applications. The model of newborn screening and MS in medicine may be useful in developing other applications that go beyond newborns and inherited metabolic disease. As MS continues to expand in clinical chemistry, it is clear that two features will drive its success. These features are excellent selectivity and multiple analyte or profile analysis; features recognized in the 1950s and remain true today.

Determination of the reference range of endogenous plasma carnitines in healthy adults.

BACKGROUND: l-carnitine is an endogenous substance, vital in the transport of fatty acids across the inner mitochondrial membrane for oxidation. Disturbances in carnitine homeostasis can have a significant impact on human health; therefore, it is critical to define normal endogenous concentrations for l-carnitine and its esters to facilitate the diagnosis of carnitine deficiency disorders. This study was conducted to determine the normal concentrations of a number of carnitines in healthy adults using three analytical methods. The impact of age and gender on carnitine concentrations was also examined. METHODS: Blood samples were collected from 60 healthy subjects of both genders and various ages. Plasma samples were analysed for endogenous carnitine concentrations by radioenzymatic assay, high-performance liquid chromatography and electrospray tandem mass spectrometry. RESULTS: Precision and accuracy of results obtained for each assay were within acceptable limits. Average endogenous concentrations obtained from the three analytical methods in this study were in the range of 38-44, 6-7 and 49-50 mumol/L for l-carnitine, acetyl-l-carnitine and total carnitine, respectively. Comparison of results between the genders indicated that males had significantly higher endogenous plasma l-carnitine and total carnitine concentrations than females. Age was found to have no impact on plasma carnitine concentrations. CONCLUSION: These results are useful in the evaluation of biochemical or metabolic disturbances and in the diagnosis and treatment of patients with carnitine deficiency.

Carnitine treatment inhibits increases in cerebral carnitine esters and glutamate detected by mass spectrometry after hypoxia-ischemia in newborn rats.

BACKGROUND AND PURPOSE: Cerebral ischemic insults disrupt normal respiratory activity in mitochondria. Carnitine plays an essential role in mitochondrial metabolism and in modulating excess acyl-coenzyme A (acyl-CoA) levels. The effects of cerebral ischemia on carnitine metabolism are not well understood, although the newborn may be particularly vulnerable to carnitine deficiency. We used a newborn rat model of hypoxia-ischemia (HI) to test the hypothesis that HI alters acyl-CoA:CoA homeostasis and that this effect can be prevented by treatment with carnitine. METHODS: A total of 120 postnatal day 7 rats were subjected to 70 minutes of HI after treatment with 16 mmol/kg intraperitoneal l-carnitine or diluent. Carnitine, acylcarnitines, and excitatory amino acids were measured by mass spectrometry, and carnitine acetyl transferase activity, superoxide, and levels of the mitochondrial phospholipid cardiolipin (CL) were measured at 2- and 24-hour recovery. RESULTS: HI and hypoxia were associated with a significant increase in the ratio of acyl-CoA:CoA, which was prevented by treatment with carnitine. Carnitine treatment also prevented increases in glutamate, glycine, superoxide, and decrease of CL. CONCLUSIONS: Carnitine metabolic pathways are compromised in HI and hypoxia. The protective effect of carnitine treatment on HI injury may be attributable to maintaining mitochondrial function.

A biochemical perspective on the use of tandem mass spectrometry for newborn screening and clinical testing.

The first newborn screen was a clinical test to detect a disorder of the biochemistry of the amino acid, phenylalanine. This disorder, known as phenylketonuria, produces profound mental retardation if not detected and treated early in life. Early screening programs relied on inexpensive population screening techniques that have all but been replaced by more accurate analytical methods such as tandem mass spectrometry (MS/MS). MS/MS enables a multianalyte approach for detecting biochemical disorders such that a metabolic profile is obtained rather than a single analyte measurement. The metabolic profile has clearly shown improvements in the detection of diseases such as phenylketonuria and several new disorders arising from errors in fatty acid oxidation and organic acid metabolism. MS/MS is a powerful tool for accessing the metabolic status of a newborn and can detect both inborn metabolic errors as well as examine the effect of acquired diseases or pharmacologic intervention on intermediary metabolism.

Tetrahydrobiopterin and nitric oxide synthase dimer levels are not changed following hypoxia-ischemia in the newborn rat.

The effect of hypoxia-ischemia on the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (BH4) and changes in the enzyme dimer state have not previously been studied. Cell-based studies have demonstrated the regulation of nitric oxide (NO) synthesis by intracellular BH4 levels. Activation of NOS requires two NOS polypeptides to form a homodimer. Dimerization results in the creation of high-affinity binding sites for BH4 and L-arginine. Our previous studies have indicated that nNOS activity falls 2 h post-hypoxia-ischemia in the immature rodent model. Thus, the objective of this study was to determine whether changes in nNOS dimeric state could be responsible for the decrease in nNOS activity. Using the immature rat model of HI in conjunction with LT-PAGE and Western blot analysis, we determined the effect of HI on NOS dimer state in hippocampus and cortex and the effects of pharmacologic modulation of NO levels during HI on dimer formation. Using high-performance liquid chromatography (HPLC) and electrospray tandem mass spectrometry (MS-MS), we measured BH4 and L-arginine levels respectively after HI under the same conditions. We found minimal or no changes in either BH4 levels or NOS dimer state at 2 h, 24 h and 7 day recovery from HI on postnatal day 7. In contrast, L-arginine levels were transiently increased in the hypoxic ischemic hemisphere. Thus, our data suggest that the previously described decrease in NOS activity after HI is not associated with depletion of the cofactor BH4, L-arginine substrate or changes in the NOS enzyme dimer state.

Impact of haemodialysis on individual endogenous plasma acylcarnitine concentrations in end-stage renal disease.

BACKGROUND: Patients with end-stage renal disease (ESRD) undergoing long-term haemodialysis exhibit low L-carnitine and elevated acylcarnitine concentrations. This study evaluated endogenous concentrations of an array of acylcarnitines (carbon chain length up to 18) in healthy individuals and ESRD patients receiving haemodialysis, and examined the impact of a single haemodialysis session on acylcarnitine concentrations. METHODS: Blood samples were collected from 60 healthy subjects and 50 ESRD patients undergoing haemodialysis (pre- and post-dialysis samples). Plasma samples were analysed for individual acylcarnitine concentrations by electrospray MS/MS. RESULTS: Of the 31 acylcarnitines, 29 were significantly (P<0.05) elevated in ESRD patients compared with healthy controls; in particular, C5 and C8:1 concentrations were substantially elevated. For acylcarnitines with a carbon chain length less than eight, plasma acylcarnitine concentrations decreased significantly over the course of a single dialysis session; however, post-dialysis concentrations invariably remained significantly higher than those in healthy subjects. Dialytic removal of acylcarnitines diminished once the acyl chain length exceeded eight carbons. CONCLUSIONS: The accumulation of acylcarnitines during long-term haemodialysis suggests that removal by haemodialysis is less efficient than removal from the body by the healthy kidney. Removal is significantly correlated to acyl chain length, most likely due to the increased molecular weight and lipophilicity that accompanies increased chain length.

Steroid profiling by tandem mass spectrometry improves the positive predictive value of newborn screening for congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is primarily caused by 21-hydroxylase deficiency and leads to an accumulation of 17-hydroxyprogesterone and reduced cortisol levels. Newborn screening for CAH is traditionally based on measuring 17-hydroxyprogesterone by different immunoassays. Despite attempts to adjust cutoff levels for birth weight, gestational age, and stress factors, the positive predictive value for CAH screening remains less than 1%. To improve this situation, we developed a method using liquid chromatography-tandem mass spectrometry to measure 17-hydroxyprogesterone, androstenedione, and cortisol simultaneously in blood spots. A total of 1222 leftover blood spots from six different screening programs using different immunoassays (fluorescent immunoassay and ELISA) were reanalyzed in a blinded fashion by liquid chromatography-tandem mass spectrometry. Thirty-one samples were from babies with CAH, 190 had yielded false-positive results by immunoassay, and the remaining 1001 samples were from babies with normal screening results. Steroid profiling allowed for an elimination of 169 (89%) of the false-positive results and for an improvement of the positive predictive value from the reported 0.5 to 4.7%. Although this method is not suitable for mass screening due to the length of the analysis (12 min), it can be used as a second-tier test of blood spots with positive results for CAH by the conventional methods. This would prevent unnecessary blood draws, medical evaluations, and stress to families.

Gestational age and age at sampling influence metabolic profiles in premature infants.

OBJECTIVE: To describe the influence that gestational age and chronological age have on amino acid and acylcarnitine profiles in an at-risk population of premature infants. METHODS: Metabolic profiles (15 amino acids and 35 acylcarnitines) were obtained by using standard newborn techniques on infants born between 23 and 31 completed weeks of gestation. The profiles were drawn within the first 24 hours after birth and on approximately days 7, 28, and 42 of life or at discharge. A single, central, contract laboratory analyzed and managed the samples. RESULTS: We studied 995 patients; none was subsequently diagnosed with an inborn error of metabolism. Of the 3579 samples, there were 257 (7.2%) amino acid or acylcarnitine alerts reported in 214 infants (21.5% of infants studied). Both gestational age and postbirth chronological age significantly influenced the metabolic profile. Twenty-nine percent of infants at 23 to 26 weeks' gestational age had an abnormal metabolic profile compared with 17% of infants at 29 to 31 weeks' gestational age (P < .01). On the day of birth, 12% of the profiles were abnormal compared with 2% on day 28 (P < .01). The highest rate of abnormal values occurred on day 7 in the infants 23 to 26 weeks' gestational age (21%). CONCLUSIONS: These results demonstrate the complexity of understanding the impact of immaturity and disease on metabolic profiles used to screen for inborn errors of metabolism. Our data provide reference values for studies aimed at better understanding metabolism in preterm infants.

Microsample analyses via DBS: challenges and opportunities.

The use of DBS is an appealing approach to employing microsampling techniques for the bioanalysis of samples, as has been demonstrated for the past 50 years in the metabolic screening of metabolites and diseases. In addition to its minimally invasive sample collection procedures and its economical merits, DBS microsampling benefits from the very high sensitivity, selectivity and multianalyte capabilities of LC-MS, which has been especially well demonstrated in newborn screening applications. Only a few microliters of a biological fluid are required for analysis, which also translates to significantly reduced demands on clinical samples from patients or from animals. Recently, the pharmaceutical industry and other arenas have begun to explore the utility and practicality of DBS microsampling. This review discusses the basis for why DBS techniques are likely to be part of the future, as well as offering insights into where these benefits may be realized.

Metabolomic profiling of fatty acid and amino acid metabolism in youth with obesity and type 2 diabetes: evidence for enhanced mitochondrial oxidation.

OBJECTIVE: We compared acylcarnitine (AcylCN) species, common amino acid and fat oxidation (FOX) byproducts, and plasma amino acids in normal weight (NW; n = 39), obese (OB; n = 64), and type 2 diabetic (n = 17) adolescents. RESEARCH DESIGN AND METHODS: Fasting plasma was analyzed by tandem mass spectrometry, body composition by dual energy X-ray absorptiometry and computed tomography, and total-body lipolysis and substrate oxidation by [(2)H(5)]glycerol and indirect calorimetry, respectively. In vivo insulin sensitivity (IS) was assessed with a 3-h hyperinsulinemic-euglycemic clamp. RESULTS: Long-chain AcylCNs (C18:2-CN to C14:0-CN) were similar among the three groups. Medium- to short-chain AcylCNs (except C8 and C10) were significantly lower in type 2 diabetes compared with NW, and when compared with OB, C2-, C6-, and C10-CN were lower. Amino acid concentrations were lower in type 2 diabetes compared with NW. Fasting lipolysis and FOX were higher in OB and type 2 diabetes compared with NW, and the negative association of FOX to C10:1 disappeared after controlling for adiposity, Tanner stage, and sex. IS was lower in OB and type 2 diabetes with positive associations between IS and arginine, histidine, and serine after adjusting for adiposity, Tanner stage, and sex. CONCLUSIONS: These metabolomics results, together with the increased rates of in vivo FOX, are not supportive of defective fatty acid or amino acid metabolism in obesity and type 2 diabetes in youth. Such observations are consistent with early adaptive metabolic plasticity in youth, which over time-with continued obesity and aging-may become dysfunctional, as observed in adults.

Metabolomic profiling of amino acids and ß-cell function relative to insulin sensitivity in youth.

CONTEXT: In longitudinal studies of adults, elevated amino acid (AA) concentrations predicted future type 2 diabetes mellitus (T2DM). OBJECTIVE: The aim of the present investigation was to examine whether increased plasma AA concentrations are associated with impaired ß-cell function relative to insulin sensitivity [i.e. disposition index (DI)], a predictor of T2DM development. DESIGN, SETTING, AND PARTICIPANTS: Metabolomic analysis for fasting plasma AAs was performed by tandem mass spectrometry in 139 normal-weight and obese adolescents with and without dysglycemia. First-phase insulin secretion was evaluated by a hyperglycemic (∼225 mg/dl) clamp and insulin sensitivity by a hyperinsulinemic-euglycemic clamp. DI was calculated as the product of first-phase insulin and insulin sensitivity. RESULTS: DI was positively associated with branched-chain AAs (leucine/isoleucine and valine; r = 0.27 and 0.29, P = 0.001), neutrally transported AAs (phenylalanine and methionine; r = 0.30 and 0.35, P < 0.001), basic AAs (histidine and arginine; r = 0.28 and 0.23, P ≤ 0.007), serine (r = 0.35, P < 0.001), glycine (r = 0.26, P = 0.002), and branched-chain AAs-derived intermediates C3, C4, and C5 acylcarnitine (range r = 0.18-0.19, P ≤ 0.04). CONCLUSION: In youth, increased plasma AA concentrations are not associated with a heightened metabolic risk profile for T2DM; rather, they are positively associated with ß-cell function relative to insulin sensitivity. These contrasting observations between adults and youth may be a reflection of developmental differences along the lifespan dependent on the combined impact of the aging process together with the impact of progressive obesity.

Altered metabolism and newborn screening using tandem mass spectrometry: lessons learned from the bench to bedside.

The use of tandem mass spectrometry (MS/MS) for screening of inherited metabolic disease in newborns has afforded many unique opportunities in the understanding of the benefits early their early detection, diagnosis and treatment. From the standpoint of the laboratory and modern analytical methods, the use of MS based analysis demonstrated that a multiple metabolite-multiple disease screen-one method approach expanded screening significantly. MS/MS and newborn screening has served as a model of one type of approach in preventative health care that has shown proven benefits. It has been nearly 20 years since the introduction of MS/MS analysis of dried blood spots from newborns. There have been many lessons learned in both the analytical approach as well as follow-up at the bedside. These lessons can be applied to future applications of MS/MS in newborn screening as well as other areas of metabolism and metabolic profiles such as that from acquired disease, environmental disease and other factors such as nutrition and age. The use of a highly specific, sensitive and multiplex platform such as MS/MS will continue to grow and experience in the newborn screening application will insure this outcome.

Technological Journey From Colorimetric to Tandem Mass Spectrometric Measurements in the Diagnostic Investigation for Phenylketonuria

Abstract Phenylalanine analysis for phenylketonuria (PKU) detection in newborn screening (NBS) was chosen as the model system to describe how advancements in laboratory technology improved laboratory performance. These advancements have made NBS programs better and have improved the health outcomes of the affected newborn through improvements in accurate early detection over the past 50 years. The most current state-of-the-art technology, tandem mass spectrometry (MS/MS), has proven that it is now the choice in almost all modern NBS facilities because it is a versatile instrument that continues to grow in its application not just for amino acid and acylcarnitine detection but for other metabolites and disorders such as lysosomal storage diseases and second-tier detection of some screen-positive results. The use of MS/MS will continue to expand, even with the anticipated introduction and expansion of molecular screening methods into NBS programs. Regarding technological advancements, the future of NBS will include even newer technologies and approaches that will enhance the detection and treatment of newborns affected by PKU and other inborn errors of metabolism.