Aminoacidopathies
Related Terms
Acylcarnitines, amino acid, aminoacidopathies, carnitine, collision cell chamber, dried blood spots, electrospray ionization, ESI, fatty acid oxidation disorder, IEM, inborn errors of metabolism, mass-to-charge ratio, mass screening, mass spectrometry, mass spectrum, MCAD, medium chain acylCoA dehydrogenase, methylmalonic and propionic aciduria, MS/MS, organic academia, peroxisomal disorders, phenylketonuria, quadrupole, succinylacetone, sudden infant death, tandem mass spectrometry, time-of-flight, tyrosine, tyrosinemia.
Background
General: Genes, made of deoxyribonucleic acid (DNA), are considered the building blocks of life because they provide instructions for all the cells in the body. They are located inside cells and control an organism's development and functions by instructing cells to make new molecules, usually proteins. Proteins are organic (carbon-containing) compounds composed of amino acids. The sequence of the amino acids in a protein is defined by a gene. Proteins are required for the growth and maintenance of the body. Amino acids are organic molecules that serve as the building blocks of proteins.
DNA is a long, thread-like (double-helix) molecule made up of large numbers of nucleotides. Nucleotides are the building blocks of DNA and are made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T). Long strands of nucleotides form nucleic acids.
Newborn screening: Newborn screening is the practice of testing newborns for certain harmful or potentially fatal disorders that may not be otherwise apparent at birth. These simple tests may determine whether newborns have certain conditions that could eventually cause health problems. Even though these conditions are considered rare and most babies are given a clean bill of health, early diagnosis and proper treatment may make the difference between potential, lifelong complications and healthy development. For example, newborn screening is used to detect a form of hearing impairment. Early detection allows for early treatment, which may decrease hearing loss.
Newborn screening programs for inherited diseases are well established in many developed countries; hence, dried blood spots on screening cards are collected from newborns for early detection of diseases and improvement of their health. The introduction of tandem mass spectrometry (MS/MS) in the 1990s by Millington and coworkers for population-based newborn screening has helped healthcare providers detect an increased number of metabolic disorders. MS/MS technology may assist in detecting metabolic disorders during the neonatal period (first 28 days of life) that previously were diagnosed only after symptoms had developed. Hence, MS/MS has been used for several years in children suspected of having inborn errors of metabolism (IEM). IEM includes a large group of genetic diseases characterized by illnesses that occur when the body is unable to process fats (lipids), proteins, sugars (carbohydrates), or nucleic acids properly. Most metabolic disorders are caused by genetic mutations (changes in genes) that result in missing or dysfunctional enzymes that are needed for the cell to perform metabolic processes.
Mass spectrometry: Mass spectrometry (MS) is an analytical technique used for measuring the molecular mass of a sample to identify the chemical composition of a compound present in the sample. Molecular mass is the mass of a molecule relative to the mass of a standard atom. The sample used for MS may be body fluids such as blood, serum, saliva, urine, etc. or tissue samples such as blood cells. MS is based on the chemical breakdown of a sample into charged ions/particles, which are then separated based on the charge and mass of the particles. The separated particles are later identified using a detector.
Electrospray ionization (ESI) is a technique in MS used to produce ions (charged particles) because ions are easier to manipulate than neutral molecules. In ESI, the sample solution containing the target compound is dissolved in a large amount of solvent that flows through a source chamber to form droplets. The droplets carry charge and highly charged analyte molecules are formed when the solvent in the solution vaporizes.
Tandem mass spectrometry (MS/MS) represents two mass spectrometers joined by a fragmentation chamber/collision cell that may break a molecule into fragments (pieces). Since there are two mass spectrometers, the abbreviation for tandem mass spectrometry is MS/MS. It is used to provide structural information about a compound by fragmenting specific sample ions inside the mass spectrometer and identifying the resulting fragment ions. A mass spectrometer is an instrument used in MS that may measure the masses of target molecules. A MS/MS is needed because a blood sample contains many compounds of which only a few compounds like amino acids or fatty acids are utilized to screen the newborns for diseases. MS/MS helps to screen only the compounds that have diagnostic significance, i.e., the compounds that may help to make a diagnosis.
Uses: MS/MS enables the sensitive and quantitative analysis of many ionic compounds such as acylcarnitines and amino acids in a single process that is taken from a single punch of a dried-blood spot. Carnitine is an ammonium compound, which has a key role in the transportation system of fats in and out of the mitochondria, the cell's energy factory. In humans, carnitines are synthesized in the liver, kidney, and brain and are actively transported to other areas of the body. It is called acylcarnitine when a fat molecule is attached to a carnitine. An acylcarnitine is known or determined by the size of the fat molecule attached to it.
The MS/MS test helps to screen newborns with inborn errors of metabolism (IEM) in the categories of fatty acid oxidation disorders, organic acidemias, and aminoacidopathies. Fatty acid oxidation disorder is a genetic metabolic deficiency wherein the body is unable to breakdown (oxidize) fatty acids to make energy because an enzyme involved in the process is either absent or does not work. Organic acidemia, also known as organic aciduria, is a group of metabolic disorders that interferes with the normal amino acid metabolism, leading to the buildup of acids in abnormal amounts. This causes damage to many organs, especially the central nervous system. Aminoacidopathies are a type of inborn error of metabolism where there is an enzyme defect that blocks the body's ability to metabolize certain proteins (amino acid). All the categories of diseases listed, which have been associated with sudden death in infancy, may be prevented or treated effectively by the early identification of these diseases or their causes with the use of MS/MS.
Methods
General: Mass spectrometry (MS) is an analytical technique used for measuring the molecular mass of a sample using a mass spectrometer. Molecular mass is the mass of a molecule relative to the mass of a standard atom. A mass spectrometer creates charged particles called ions from molecules and analyzes those ions to provide information about the molecular weight/mass of the compound and its chemical structure. A mass spectrometer is divided into three areas, namely the ionization source, the analyzer, and the detector.
A tandem mass spectrometer is an instrument used in MS that has more than one analyzer, in practice usually two. The two analyzers are separated by a fragmentation chamber/collision cell into which an inert gas (e.g., argon, xenon) is admitted to collide with the selected sample ions and bring about their breakdown (fragmentation). The fragmented ions are passed through the two analyzers, which may be of the same or of different types. The most common combinations being: quadrupole-quadrupole or quadrupole-time-of-flight analyzers, which are explained below. The major steps involved in electrospray tandem mass spectrometry are described below.
First mass spectrometer: The first step involves the introduction of the sample into the ionization source of the first mass spectrometer. Electrospray ionization (ESI) is a technique in MS used to produce ions because ions are easier to manipulate than neutral molecules, thereby facilitating the identification of the target molecule/analyte in the sample. In ESI, the sample solution containing the analyte is dissolved in a large amount of solvent that flows through a source chamber to form droplets. The droplets carry charge and highly charged analyte molecules are formed when the solvent in the solution vaporizes.
Mass analyzer: The ions then move into the analyzer region of the first mass spectrometer where the ions are separated and weighed according to their mass-to-charge ratios. The mass-to-charge ratio is the physical quantity that may be calculated by dividing the charge of the substance/analyte by the mass of the same substance. There are a number of mass analyzers available and some of them include time-of-flight (TOF) analyzers, quadrupoles, etc. TOF analyzers use an electric field to filter ions based on their mass-to-charge ratio. The quadrupole mass analyzer consists of four circular rods that are parallel to each other and facilitate the filtering of sample ions based on their mass-to-charge ratio.
Collision cell chamber: The ions produced and analyzed in the first mass spectrometer then enter the collision cell chamber. The ions are broken into smaller pieces using an inert gas (e.g. argon, xenon), which collides with the selected sample ions and brings about their fragmentation to sort out the ions needed by the researchers for the diagnosis of disease.
Second mass spectrometer:
The sorted or selected ions are then introduced into the second mass spectrometer for further analysis. The selected analyte is separated based on its mass-to-charge ratios, which is similar to the process of analysis in the first mass spectrometer.
Detector: The detector in the mass spectrometer monitors the ion current and amplifies (increases) it. There are many types of detectors and most of them produce an electronic signal when struck by an ion. The signal is transmitted to the data system (computer programs) and is stored in the form of mass spectra. A mass spectrum is commonly presented as a vertical bar graph, wherein each bar represents an ion having a specific mass-to-charge ratio (m/z) and the amount of the ion is indicated by the length of the bar. The m/z value of an ion is equivalent to mass itself because most of the ions formed in the mass spectrometer have a single charge. Hence, this method facilitates the accurate identification of the target molecule. Thus, MS/MS helps to identify the compound that is responsible for causing a disorder in newborns and assists in the early identification of the disorder allowing the treatment to begin at an early stage for better treatment outcomes.
Research
Electrospray tandem mass spectrometry (MS/MS) may assist in diagnosing several metabolic disorders. Several studies are being conducted to improve MS/MS as well as detect more metabolic disorders in newborns using MS/MS. Some of the studies are described below.
Methylmalonic and propionic aciduria: Both of these diseases are types of organic aciduria, which is a group of metabolic disorders that interferes with the normal amino acid metabolism, leading to a buildup of acids in abnormal amounts. This causes damage to many organs, especially the central nervous system. Methylmalonic and propionic aciduria have been found due to the decreased function of enzymes, methylmalonyl-CoA mutase and propionyl-CoA carboxylase, respectively. The screening of newborns with electrospray tandem mass spectrometry (MS/MS) may decrease the incidence of deaths thanks to early detection and the treatment of diseases in affected children.
Peroxisomal disorders: Peroxisomal disorders are a group of metabolic diseases that have defective peroxisomes, which is an organelle (a specialized organ within a cell) that helps in cell function such as the oxidation of fatty acids. The identification of fatty acids (hexacosanoic acid) is the basis for the detection of these disorders; hence, researchers have used MS/MS along with ultra performance liquid chromatography (UPLC) to detect these disorders. UPLC is a type of analytical technique used in a laboratory to separate, identify, and quantify (count) compounds. The degree of separation is increased by forcing a solvent under pressure through a densely packed adsorbent/absorptive material. Initial results of the studies show promise; however, further research is required to examine the benefits and drawbacks of the combined technique.
Tyrosinemia type 1: Tyrosinemia is a type of genetic disorder wherein an elevated level of the amino acid tyrosine is found in the blood due to the decreased amount of the enzyme fumarylacetoacetate hydrolase. Symptoms usually appear in the first few months of life and include failure to gain weight or grow at the expected rate (failure to thrive) and may lead to liver and kidney failure. Researchers have conducted studies using MS/MS and UPLC to identify this disorder by determining the level of succinylacetone, a metabolite elevated in tyrosinemia type 1.
Implications
General: Electrospray tandem mass spectrometry (MS/MS) may assist in detecting metabolic disorders during the neonatal period (first 28 days of life) that previously were diagnosed only after symptoms developed later in life. MS/MS is a technique or type of mass spectrometry (MS) used for measuring the molecular mass of a sample; it helps to identify the chemical composition of a compound present in the sample. MS/MS has been used for several years in children suspected of having inborn errors of metabolism (IEM) using just a single blood drop. IEM include a large group of genetic diseases characterized by illnesses that occur when the body is unable to process fats (lipids), proteins, sugars (carbohydrates), or nucleic acids properly.
Generally, in metabolic disorders, specific enzymes that facilitate the breakdown of amino acids or that are needed for converting fat to energy do not work or are absent. This prevents a compound from breaking down/being metabolized and thus it accumulates in the blood and tissues of the body. The accumulated compound is toxic or poisonous, causing damage to different organs of the body. MS/MS detects the levels of this toxic compound and helps in the diagnosis of disorders.
In newborns, MS/MS enables medical professionals to count the number of many compounds, such as acylcarnitines and amino acids, in a single process. Carnitine is an ammonium compound that has a key role in the transportation system of fats in and out of the mitochondria, the cell's energy factory. In humans, carnitines are synthesized in the liver, kidney, and brain and actively transported to other areas of the body. It is called as an acylcarnitine when a fat molecule is attached to a carnitine. An acylcarnitine is known or determined by the size of the fat molecule attached to it. The acylcarnitine is classified as a short, medium, or long chain of fats. For example, in a metabolic disorder called medium chain acylCoA dehydrogenase deficiency (MCADD), the fat attached to carnitine is a medium-sized eight-carbon fatty acid known as octanoylcarnitine.
MS/MS helps to screen newborns with inborn errors of metabolism (IEM) in the categories of fatty acid oxidation disorders, organic acidemias, and aminoacidopathies, which are explained below. All these categories of diseases, which are known to cause sudden death in infancy, may be prevented or treated effectively by the early identification of these diseases by MS/MS.
Aminoacidopathies: Aminoacidopathies are a type of inborn error of metabolism (IEM) where there is an enzyme defect that inhibits the body's ability to metabolize certain proteins (amino acid).
Fatty acid oxidation disorders: Fatty acid oxidation disorder is a genetic metabolic deficiency wherein the body is unable to breakdown (oxidize) fatty acids to make energy because an enzyme involved in the process is either absent or is nonfunctional.
Maple syrup urine disease (MSUD): MSUD is a disease caused by the defective metabolism of amino acids such as leucine, isoleucine, and valine due to low levels of an enzyme called branched-chain alpha-keto acid dehydrogenase (BCKD) in the blood. This results in amino acids being present in the blood and urine in high concentrations. Some signs of the disease include feeding difficulties, physical and mental retardation, and a urine odor similar to that of maple syrup. Neonatal death is also common.
Medium chain acylCoA dehydrogenase deficiency (MCADD): MCADD is diagnosed based on an elevated level of the fat attached to carnitine known as octanoylcarnitine. It is one of the most common fatty acid oxidation disorders, presenting as acute episodes triggered by prolonged fasting for more than 12-16 hours, with hypoglycemia (reduced blood sugar level), vomiting, and fatigue or weariness. This may progress to seizures, coma, or even sudden death and usually presents before the age of three years.
Organic acidemia: Organic acidemia, also known as organic aciduria, is a group of metabolic disorders that interferes with the normal amino acid metabolism, leading to the buildup of acids in abnormal amounts. This causes damage to many organs, especially the central nervous system. One of the examples of this disorder is explained below.
Phenylketonuria (PKU): Newborns with PKU lack the enzyme phenylalanine hydroxylase that converts the amino acid phenylalanine to tyrosine. If left untreated, this leads to excessive buildup of phenylalanine and its breakdown products in the blood that are toxic to the brain, causing brain damage and progressive mental retardation.
Sudden infant death syndrome (SIDS): SIDS is the sudden and unexplained death of an infant younger than one year of age. It may strike without warning, usually in a seemingly healthy infant. It is a leading cause of death among infants who are one month to one year old; it claims the lives of about 2,500 infants each year in the United States. It is most common, however, between the ages of two and four months. Although multiple factors may be involved, some studies have suggested that fatty acid metabolism disorders may be responsible for cases of sudden and unexpected death in infancy.
Advantage of MS/MS: Due to the large increase in the number of inherited metabolic disorders detectable in the newborn period, MS/MS screening improves the possibility of early detection of disorders before the initiation of symptoms and helps to start the treatment while the infant is still healthy. MS/MS also assists in defining the range of clinical diseases related to these disorders and thereby may reduce deaths due to these disorders in the affected children. MS/MS also provides rapid results (within one to two minutes), and therefore may be used to screen large numbers of samples in newborn screening programs.
Limitations
The diseases screened using tandem mass spectrometry (MS/MS) are rather rare and vary in clinical severity. Also, the screening's clinical utility or usefulness and cost-effectiveness have not yet been fully established. Hence, further careful data collection regarding the sensitivity of MS/MS screening and outcomes of patients in whom disorders are detected is required.
The cost of MS/MS itself is expensive and may be impractical to use in many countries for mass screening programs. Also, tandem mass spectrometry requires experts (trained personnel) to perform the analyses and interpret the large amount of clinical data produced from the analysis of a blood sample. Hence, to get accurate results, people have to be trained, which again increases cost and time.
MS/MS is a useful tool to detect specific metabolic conditions through newborn screening. False negative and false positive results, however, do occur. Close coordination between the laboratories performing the screening is needed to determine screening parameters and to adjust cut-off levels to reduce false-positive and false-negative results. A false positive is a positive test result in spite of the true absence of a disease/disorder and a false negative is a negative test result in spite of the true presence of a disease/disorder.
Future research
General: Tandem mass spectrometry (MS/MS) is a powerful tool that expands newborn screening for many diseases. Along with other types of mass spectrometry, it may be used to analyze other treatable disorders or to improve existing tests so that more accurate and more comprehensive tests may be available in future. This may lead to the further development of new therapies along with the improvement of existing treatments, which may benefit future generations.
Maple syrup urine disease (MSUD): Researchers are conducting studies analyzing the results of newborn screening with electrospray tandem mass spectrometry for inborn errors of metabolism, such as MSUD. MSUD is a disorder due to low levels of an enzyme called branched-chain alpha-keto acid dehydrogenase (BCKD) in the blood. They have found that newborns with the intermediate form of MSUD cannot be diagnosed with this method, thus suggesting that partial enzyme deficiencies may not be detected using MS/MS. Hence, further research is required to improve MS/MS detection of mild or intermediate forms of metabolic disorders.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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