Urine Screening For Metabolic Disorders PDF

URINE SCREENING FOR METABOLIC DISORDERS MT104 AUBF LAWRENCE R. SANTIAGO, RMT Introduction  Many abnormal urinalysis results are usually related to metabolic rather than renal disease.  Positive screening tests can then be followed up with more sophisticated procedures performed...

URINE SCREENING FOR METABOLIC DISORDERS MT104 AUBF LAWRENCE R. SANTIAGO, RMT Introduction  Many abnormal urinalysis results are usually related to metabolic rather than renal disease.  Positive screening tests can then be followed up with more sophisticated procedures performed in other sections of the laboratory  The need to perform additional tests may be detected by the observations of alert laboratory personnel when performing the routine analysis or from observations of abnormal specimen color and odor by nursing staff and patients.  In other instances, clinical symptoms and family histories are the deciding factors Overflow versus Renal Disorders  Overflow disorders: disruption of a normal metabolic pathway that causes increased plasma concentrations of the nonmetabolized substances.  1. Override the reabsorption ability of the renal tubules  2. Not normally reabsorbed from the filtrate (present in minute amts)  Renal Disorders: caused by malfunctions in the tubular reabsorption mechanism Overflow - continued  Metabolic disturbances that produce overflow: involve Protein, Fat, and Carbohydrate metabolism  Disruption of enzyme function can be caused by failure to inherit the gene to produce a particular enzyme, referred to as an inborn error of metabolism (IEM) Newborn Screening Tests  Used primarily to detect and monitor IEMs  Newborn screening tests in the Philippines expanded from 6 to 28 with the initial 6 NBS test include:  1. Congenital Hyperthyroidism  2. Phenylketonuria  3. Congenital adrenal hyperplasia  4. Glucose-6 phosphate dehydrogenase (G6PD) deficiency  5. Galactosemia  6. Maple Syrup Disease  1. Congenital Hyperthyroidism: - excessive thyroid hormones that could lead to growth failure and intellectual disability.  2. Phenylketonuria - an inherited disorder where children cannot breakdown the amino acid phenylalanine, which can lead to brain damage.  3. Congenital adrenal hyperplasia - a group of genetic disorders affecting the adrenal glands – structures that produce vital hormones. Babies with CAH lack one enzyme to make these vital hormones and are at risk of adrenal crisis, a life-threatening condition.  4. Glucose-6 phosphate dehydrogenase (G6PD) deficiency - an inherited condition where babies lack the G6PD enzyme. This can result in hemolytic anemia, which can be fatal when not properly treated.  5. Galactosemia - the inability to metabolize galactose, a type of sugar commonly found in breastmilk. This condition can be life- threatening if not treated early.  6. Maple Syrup Disease - Like phenylketonuria, babies with MSUD cannot metabolize specific proteins. Infants with this condition have maple-smelling urine. Left untreated, MSUD can be fatal.  Early detection of defects is vital to prevent buildup of unmetabolized toxic food ingredients  Levels of these substances are elevated more rapidly in blood than urine  therefore, blood collected by infant heel puncture is initially tested Amino Acid Disorders PHENYLALANINE-TYROSINE DISORDERS Phenylketonuria  The most well known of the aminoacidurias  estimated to occur in 1 of every 10,000 to 20,000 births  if undetected, results in severe mental retardation  first identified in Norway by Ivan Følling in 1934, when a mother with other mentally retarded children reported a peculiar mousy odor to her child’s urine  increased amounts of keto acids including phenylpyruvate  normal conversion of phenylalanine to tyrosine is disrupted Continued  Interruption of the pathway also produces children with fair complexions—even in dark-skinned families— owing to the decreased production of tyrosine and its pigmentation metabolite melanin  PKU is caused by failure to inherit the gene to produce the enzyme phenylalanine hydroxylase  The gene is inherited as an autosomal recessive trait with no noticeable characteristics or defects exhibited by heterozygous carriers  Once discovered, dietary changes that eliminate phenylalanine, a major constituent of milk, from the infant’s diet can prevent excessive buildup of serum phenylalanine  thereby avoiding damage to the child’s mental capabilities.  As the child matures, alternative pathways of phenylalanine metabolism develop, and dietary restrictions can be eased.  Many products that contain large amounts of phenylalanine, such as aspartame, now features warning labels for people with PKU.  Initial screening for PKU goes undetected in the urinalysis lab as the increased blood levels of phenylanaline must occur before urinary excretion of phenylpyruvic acid (takes 2 to 6 weeks)  Blood is collected 24 hours after birth  studies have shown that in many cases phenylalanine can be detected as early as 4 hours after birth and if cutoff values are lowered from 4 mg/dL to 2 mg/dL.  More girls than boys escape detection of PKU during early tests because of slower rises in blood phenylalanine levels. Courtesy of Reynan P. Borja, RMT Courtesy of Reynan P. Borja, RMT Courtesy of Reynan P. Borja, RMT Courtesy of Reynan P. Borja, RMT Courtesy of Reynan P. Borja, RMT Phenylketonuria *Guthrie test is a semiquantitative, bacterial inhibition assay for phenylalanine that uses the ability of phenylalanine to facilitate bacterial growth(Bacilus subtilis) in a culture medium with an inhibitor (ꞵ-2- thienylalanine). + for Phenylketonuria = GROWTH provide false negative results due to the infant not being at least 24 hours old, which ensures adequate time for enzyme and amino acid levels to develop, and due to the sample not being taken before the administration of antibiotics or transfusion of blood or blood products. Phenylketonuria U.S. Food and Drug Administration (FDA) approved Kuvan (sapropterin TREATMENT dihydrocholoride), the first drug to The goal of PKU treatment is to maintain help manage PKU. The drug helps the blood level of phenylalanine between reduce phenylalanine levels by increasing the activity of the PAH 2 and 10 mg/dL (120–600 umol/L). Some enzyme. phenylalanine is needed for normal growth, so a diet that has some Kuvan is effective only in patients phenylalanine but in much lower amounts who have some PAH activity and who continue to follow a phenylalanine- than normal is the recommended restricted diet and have their treatment. High-protein foods, such as phenylalanine levels monitored. meat, fish, poultry, eggs, cheese, and milk, are avoided. Instead, calculated amounts of cereals, starches, fruits, and vegetables, along with a milk substitute, are usually recommended.  Urine testing using ferric chloride may be used as a followup test to ensure proper dietary control in previously diagnosed cases and as a means of monitoring the dietary intake of pregnant women known to lack phenylalanine hydroxylase  Urine tests for phenylpyruvic acid are based on the ferric chloride reaction performed by tube test. The addition of ferric chloride to urine containing phenylpyruvic acid produces a permanent blue-green color Tyrosyluria  The accumulation of excess tyrosine in the plasma (tyrosinemia)  tyrosine metabolism disorders may result from either inherited or metabolic defects.  two reactions are directly involved in the metabolism of tyrosine  urine may contain excess tyrosine or its *degradation products  *p-hydroxyphenylpyruvic acid and p- hydroxyphenyllactic acid Continued  Most frequently seen is a transitory tyrosinemia in premature infants, which is caused by underdevelopment of the liver function required to produce the enzymes necessary to complete the tyrosine metabolism.  Acquired severe liver disease also produces tyrosyluria resembling that of the transitory newborn variety and, of course, is a more serious condition. In both instances, rarely seen tyrosine and leucine crystals may be observed during microscopic examination of the urine sediment. 2.) Tyrosinemia TREATMENT  Diagnostic criteria include an elevated  Low-protein diet  Nitisinone (NTBC)- prevents the tyrosine level using MS/MS coupled with a formation of maleylacetoacetic confirmatory test for an elevated level of acid and fumarylacetoacetic acid, the abnormal metabolite which can be converted to succinylacetone-toxin that damages the succinylacetone liver and kidneys.  full or partial liver transplant  Urine testing for tyrosyluria using nitroso- Since nitisinone’s first use for naphthol : 1.) Place five drops of urine in tyrosinemia in 1991, it has a tube. 2.) Add 1 mL of 2.63N nitric acid. replaced liver transplantation 3.) Add one drop of 21.5% sodium nitrite. as the first-line treatment for 4.) Add 0.1 mL 1-nitroso-2-napthol. 5. Mix. this rare condition. 6. Wait 5 minutes 7. Observe for an orange-red color, indicating tyrosine metabolites.  Ferric Chloride tube test: Transient green Continued  Hereditary disorders in which enzymes required in the metabolic pathway are not produced present serious and often fatal conditions that result in both liver and renal tubular disease producing a generalized aminoaciduria.  Based on the enzymes affected, the hereditary disorders can be classified into three types, all producing tyrosylemia and tyrosyluria. Tyrosinemia Type 1  produces a generalized renal tubular disorder and progressive liver failure in infants soon after birth  caused by low levels of the enzyme fumarylacetoacetate hydrolase Tyrosinemia Type 2  caused by lack of the enzyme tyrosine aminotransferase  Persons develop corneal erosion and lesions on the palms, fingers, and soles of the feet believed to be caused by crystallization of tyrosine in the cells. Tyrosinemia Type 3  caused by lack of the enzyme p- hydroxyphenylpyruvic acid dioxygenase  result in mental retardation if dietary restrictions of phenylalanine and tyrosine are not implemented. Nitroso-Napthol Test for Tyrosine Melanuria  a second metabolic pathway also exists for tyrosine.  This pathway is responsible for the production of melanin, thyroxine, epinephrine, protein, and tyrosine sulfate.  major concenern of the urinalysis laboratory is melanin  melanin: pigment responsible for the dark color of hair, skin, and eyes  Deficient production of melanin results in albinism  Increased urinary melanin darkens the urine upon exposure to air  Elevated urinary  melanin is a serious finding that indicates proliferation of the normal melanin-producing cells (melanocytes) producing malignant melanoma Continued  These tumors secrete a colorless precursor of melanin, 5,6-dihydroxyindole, which oxidizes to melanogen and then to melanin, producing the characteristic dark urine. Alkaptonuria  Alkaptonuria was one of the six original inborn errors of metabolism described by Garrod in 1902  derived from the observation that urine from patients with this condition darkened after becoming alkaline from standing at room temperature. (“alkali lover,” or alkaptonuria, was adopted)  the third major defect in the phenylalanine- tyrosine pathway and occurs from failure to inherit the gene to produce the enzyme homogentisic acid oxidase Continued  Without this enzyme, the phenylalanine-tyrosine pathway cannot proceed to completion, and homogentisic acid accumulates in the blood, tissues, and urine.  observations of brown-stained or black-stained cloth diapers and reddish-stained disposable diapers have been reported.  In later life, brown pigment becomes deposited in the body tissues (particularly noticeable in the ears). Deposits in the cartilage eventually lead to arthritis. A high percentage of persons with alkaptonuria develop liver and cardiac disorders Continued  Homogentisic acid reacts in several of the routinely used screening tests for metabolic disorders, including the ferric chloride test, in which a transient deep blue color is produced in the tube test.  A yellow precipitate is produced in the Clinitest, indicating the presence of a reducing substance.  Another screening test for urinary homogentisic acid is to add alkali to freshly voided urine and to observe for darkening of the color; however, large amounts of ascorbic acid interfere with this reaction. 4.) Alkaptonuria  Urinalysis is done to test for alkaptonuria. *Ferric Chloride test BISHOP: Black; STRASINGER: Transient deep blue *Clinitest/Benedict’s test : Yellow precipitate *Homogentisic Acid Test : 1. Place 4 mL of 3% silver nitrate in a tube. 2. Add 0.5 mL of urine. 3. Mix. 4. Add 10% NH4OH by drops. 5. Observe for black color (after the addition of base/alkali)  Treatment for alkaptonuria is high-dose vitamin C, which has been shown to decrease the buildup of brown pigment in the cartilage and may slow the development of arthritis.  Amino Acid Disorders BRANCHED-CHAIN AMINO ACID DISORDERS Courtesy of Reynan P.Borja, RMT Maple Syrup Urine Disease (MSUD)  Maple syrup urine disease (MSUD) is a lifelong and potentially life-threatening inherited metabolic disorder. Metabolic disorders cause problems with how the body breaks down food into the tiny components it uses for energy. With MSUD, the body has trouble breaking down amino acids, the building blocks of protein. People with MSUD have trouble breaking down three amino acids in particular:  Leucine.  Isoleucine.  Valine. How common is maple syrup urine disease (MSUD)?  MSUD is very rare. It affects about 1 in every 185,000 babies born worldwide.  Autosomal recessive trait  It occurs more often in populations with a small gene pool or when close relatives have children together. For example, MSUD is more common among Mennonites in the United States (1 in every 380 births), where community members often marry and have kids. It’s also more common in the Ashkenazi Jewish population (1 in every 26,000 births). Types of MSUD  Classic: Classic maple syrup urine disease is the most severe type. It’s also the most common. Symptoms usually develop within the first three days of birth.  Intermediate: This type of MSUD is less severe than classic MSUD. Symptoms typically appear in children between 5 months and 7 years old.  Intermittent: Children with intermittent MSUD develop as expected until an infection or period of stress causes symptoms to appear. People with intermittent MSUD usually tolerate higher levels of the three amino acids than those with classic MSUD.  Thiamine-responsive: This type of MSUD responds to treatment using high doses of vitamin B1 (thiamine) along with a restricted diet. With treatment, people with thiamine-responsive MSUD have a higher tolerance for the three amino acids. BRANCHED-CHAIN AMINO ACID DISORDERS 1.) Maple Syrup Urine Disease (MSUD)  absence or greatly reduced activity of the enzyme branched-chain ⍺- ketoacid decarboxylase complex, blocking the normal metabolism of the three essential branched-chain amino acids leucine, isoleucine, and valine.  characteristic maple syrup or burnt sugar odor of the urine, breath, and skin.  The result of this enzyme defect is an accumulation of the branched- chain amino acids and their corresponding ketoacids in the blood, urine, and cerebrospinal fluid (CSF) causing ACIDEMIA  Infants with MSUD seem normal at birth but, within a week, the disease progresses to cause severe mental retardation, seizures, acidosis, and hypoglycemia. If treatment is not given, the disease can lead to death.  Studies have shown that if maple syrup urine disease is detected b the 11th day, dietary regulation and careful monitoring of urinary keto acid concentrations can control the disorder 1.) Maple Syrup Urine Disease (MSUD) 2,4-dinitrophenylhydrazine (DNPH) urine screening test for MSUD Aka Brady’s Test 1. Place 1 mL of urine in a tube. 2. Add 10 drops of 0.2% 2,4-DNPH in 2N HCl. 3. Wait 10 minutes. 4. Observe for yellow turbidity or precipitate Management and Treatment  Diet Limited protein intake (meat, dairy, legumes)  Monitoring Amino acid monitoring (leucine, isoleucine, valine)  Emergency care for metabolic crises Glucose (as 10% dextrose) and insulin Nasogastric feeding tube (delivers particular nutrients) Hemodialysis Monitoring for signs of brain swelling or infection and acid buildup Is there a cure?  Since 2004, liver transplants have successfully treated people with classic MSUD. A new liver can produce the enzymes needed to break down the three amino acids. After a liver transplant, you can eat an unrestricted diet, live without symptoms, and avoid further symptoms or complications.  But you’ll still carry the gene for MSUD, which means you can pass it on to your child