Amino acids newborns

The optimization of MS/MS for amino acids in DBS was the first step in the development and validation of NBS [2]. The reason that an amino acids newborn screen was developed first rather than an AC screen was the requirement for validation of using a DBS for analysis and quantification by MS/MS. By choosing a compound that is MS friendly (i.e., positive ion, small molecule, previous MS analyses) and is also a key metabolite in NBS, it was more likely that validation of the assay would be achieved and that validation accepted. Phe is that MS-friendly metabolite used in the detection of PKU and screened for by all 50 US. states. It was likely that development of the Phe assay using MS/MS would be similar to that for ACs, since chemically speaking they are not that different in terms of molecular weight and the fact that they both have basic and acid groups. Sample extraction and preparation are also likely to be similar. So the initial attempts at an assay could mirror that used for plasma ACs. 

Tabic 1.1. Amino acids per thousand residues for standard type 1 collagen and 15 human bone collagen samples from St. Thomas Church cemetery. Samples are identified by burial numlter (Bx) followed by age midpoint for subadults and age range for adults in parentheses. For example, B17(0) refers to burial 17 who was aged as a newborn. 

There are also RMs which are prepared for a specific application and are used for validation of relevant methods. Cobbaert et al. (1999) made use of Ion Selective Electrode (ISE)-protein-based materials when evaluating a procedure which used an electrode with an enzyme-linked biosensor to determine glucose and lactate in blood. Chance et al. (1999) are involved with the diagnosis of inherited disorders in newborn children and they prepared a series of reference materials consisting of blood spotted onto filter paper and dried, from which amino-acids can be eluted and... 

Chance DH, Adam BW, Smith SJ, Alexander JR, Hillman SL, Hannon WH (1999) Validation of accuracy-based amino acid reference materials in dried-blood spots by tandem mass spectrometry for newborn screening assays. Clin Chem 45 1269-1277. 

The urea cycle is essential for the detoxification of ammonia 678 Urea cycle defects cause a variety of clinical syndromes, including a metabolic crisis in the newborn infant 679 Urea cycle defects sometimes result from the congenital absence of a transporter for an enzyme or amino acid involved in the urea cycle 680 Successful management of urea cycle defects involves a low-protein diet to minimize ammonia production as well as medications that enable the excretion of ammonia nitrogen in forms other than urea 680... 

Other conditionally essential amino acids, such as cysteine, carnitine, and glutamine, are not included because they are unstable or insoluble. Cysteine and carnitine are commonly added to PN solutions compounded for newborns. 

This in fact is what happened. Tandem MS has clearly been shown to be the only technology to screen for disorders of fatty acid oxidation and could also detect many disorders of organic acid metabolism. Tandem MS has the ability to detect both compound classes (amino acids and acylcarnitines) and after demonstrating that both classes could be prepared in the same manner, the MS/MS analysis of blood spots for newborn screening applications was born. 

A much more serious genetic disease, first described by Foiling in 1934, is phenylketonuria. Here the disturbance in phenylalanine metabolism is due to an autosomal recessive deficiency in liver phenylalanine hydroxylase (Jervis, 1954) which normally converts significant amounts of phenylalanine to tyrosine. Phenylalanine can therefore only be metabolized to phenylpyruvate and other derivatives, a route which is inadequate to dispose of all the phenylalanine in the diet. The amino acid and phenylpyruvate therefore accummulate. The condition is characterized by serious mental retardation, for reasons which are unknown. By the early 1950s it was found that if the condition is diagnosed at birth and amounts of phenylalanine in the diet immediately and permamently reduced, mental retardation can be minimized. The defect is shown only in liver and is not detectable in amniotic fluid cells nor in fibroblasts. A very sensitive bacterial assay has therefore been developed for routine screening of phenylalanine levels in body fluids in newborn babies. 

Reduced forms of folic acid are required for essential biochemical reactions that provide precursors for the synthesis of amino acids, purines, and DNA. Folate deficiency is not uncommon, even though the deficiency is easily corrected by administration of folic acid. The consequences of folate deficiency go beyond the problem of anemia because folate deficiency is implicated as a cause of congenital malformations in newborns and may play a role in vascular disease (see Folic Acid Supplementation A Public Health Dilemma). 

Dickenson JC, Rosenblum H, Hamilton PB (1965) Ion exchange chromatography of the free amino acids in the plasma of newborn infants. Pediatrics 36 2-13... 

In metabolism, more specifically newborn screening for amino acids and acyl-carnitines, we use two other types of MS/MS analyses the precursor and neutral loss scans. In both types of scan, the question is asked where did I come from In other words, a fragment of a molecule is detected that is unique or common to a particular molecule or family of structurally similar molecules. Because the instruments are linked in space rather than time, when a particular product is detected in the second mass spectrometer (MS2), the computer software knows what mass was being scanned in the first mass spectrometer (MSI). The difference between a precursor ion and neutral loss scan is the nature of the product. With a precursor ion scan, that product of interest is also an ion and it can be detected. With a neutral loss... 

In early infancy the situation is known to be quite different from that among older children. Simon noticed in 1911 that the newborn infant excreted 10 % of its total nitrogen output in the form of amino nitrogen, whereas in adults it drops to 2 % (S15). This was confirmed in later years (B4, Cll, G5) and extended to the study of individual amino acid excretion by microbiological methods (S10, Sll), by paper chromatography (S20, S21), and finally by ion exchange column chromatography (D28, D30) at the time Moore and Stein s first method of 1951 was still the only one available. 

Today, many of the older methods used historically in clinical chemistry are being replaced by methods that utilize MS. In 2013, some areas of interest in clinical chemistry include steroid and vitamin D analyses that require a high selectivity and relative sensitivity. For example, the improved selectivity is essential in the analysis of testosterone in infants due to low concentrations. Immunoassays are not sufficiently selective to provide an accurate measurement of the true concentrations of these metabolites. There are many more assays being used or that are now in development in clinical chemistry, but a discussion of this growing use of metabolic screening is too broad and too evolutionary to be included here. Therefore, this chapter focuses on those proteins and metabolites (e.g., amino acids and acylcamitines) that are part of a classic newborn screening panel. 

For new disorders, acceptance came from detecting patients with the disorder in numbers that were similar to PKU, as was the case with MCAD deficiency. When the number of newborns screened approached a half million and at least one or two very rare disorders (1 in 100,000-300, 000) could be detected, the statistics supported the continued use of the method. A comprehensive MS/MS screen that included an amino acid and acylcami-tine profile could detect a disorder in 1 infant in 2500 screened while having a false result rate equal to or less than many of the newborn screens for one disorder. 

An inability to degrade amino acids causes many genetic diseases in humans. These diseases include phenylketonuria (PKU), which results from an inability to convert phenylalanine to tyrosine. The phenylalanine is instead transaminated to phenylpyruvic acid, which is excreted in the urine, although not fast enough to prevent harm. PKU was formerly a major cause of severe mental retardation. Now, however, public health laboratories screen the urine of every newborn child in the United States for the presence of phenylpyru-vate, and place children with the genetic disease on a synthetic low-phenylalanine diet to prevent neurological damage. 

There are two pyridoxal phosphate-requiring enzymes in the homocysteine degradation pathway, which are associated with genetic diseases. In homo-cystinuria, cystathionine synthase is defective, and large amounts of homocystine are excreted in the urine. Some homocystinurics respond to the administration of large doses of vitamin B6. In cystathioninuria, cystathionase is either defective or absent. These patients excrete cystathionine in the urine. Cystathionase is often underactive in the newborns with immature livers, and cysteine and cystine become essential amino acids. Human milk protein is especially rich in cysteine, presumably to prepare the newborn for such a contingency. 

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