Clinical Chemistry - Lecture 3: Protein, Amino Acids, Aminoacidopathies

Name some nonprotein nitrogen (NPN) compounds amino acids help form.

Purines, pyrimidines, porphyrins, creatine, histamine, thyroxine, epinephrine, and coenzyme NAD.

What types of energy sources can amino acids be?

Glucogenic and ketogenic.

What are the primary aminoacidopathies listed in the lecture?

Phenylketonuria (PKU)

Tyrosinosis,

Cystinosis,

Homocystinuria,

Maple syrup urine disease (MSUD), and alkaptonuria.

What gene is mutated in PKU?

PAH gene.

What enzyme does the PAH gene encode?

Phenylalanine hydroxylase.

What is the inheritance pattern of PKU?

Autosomal recessive.

What is the frequency of PKU?

1:15,000.

Is newborn testing for PKU required in the USA?

Yes!

What diet is required for PKU patients?

A special diet with low phenylalanine for life.

Which metabolites can cause significant and permanent brain damage in PKU?

Phenylpyruvic acid, phenylpyruvate, and phenyllactic acid.

What odor is associated with PKU urine?

Musty or mousy odor.

What neurological findings are associated with PKU?

Progressive mental retardation and fetal microcephaly.

When is PKU testing performed in newborns?

On blood at day 3 after birth.

What organism is used in the Guthrie test?

Bacillus subtilis spores.

What antagonist is used in the Guthrie test?

β2-thienylalanine.

How does increased phenylalanine affect the Guthrie test?

It inhibits the action of β2-thienylalanine so spores grow.

What is the detection limit of the Guthrie test?

3 mg/dL.

What methods are replacing the Guthrie test?

HPLC and MS/MS.

What color reaction occurs with a positive Phenistix test?

Blue-gray to green.

What confirmatory methods are used for PKU?

HPLC and tandem MS.

What happens when excess tyrosine accumulates in blood?

Overflow of tyrosine into urine.

What odor is associated with tyrosinemia urine?

Cabbage-like odor.

Which mutation causes Type I tyrosinemia?

FAH gene mutation.

Which type of tyrosinemia is most severe?

Type I.

What complications occur in Type I tyrosinemia?

Severe liver disease, cirrhosis, and liver cancer risk later in life.

Which mutation causes Type II tyrosinemia?

TAT gene mutation.

What findings occur in Type II tyrosinemia?

Impaired mental development, painful skin lesions, and photophobia.

Which mutation causes Type III tyrosinemia?

HPD gene mutation.

What findings occur in Type III tyrosinemia?

Impaired mental development, seizures, and loss of balance.

What laboratory findings are seen in tyrosinemia?

Elevated tyrosine and succinylacetone in blood and tyrosine crystals in urine.

What color result is seen in the nitrosonaphthol test?

Positive red color.

What confirmatory tests are used for tyrosinemia?

MS/MS or quantitative tyrosine assay.

How do tyrosine crystals appear microscopically?

Fine silky needles arranged in sheaves or bundles in acid urine.

With what other crystals may tyrosine crystals appear?

Leucine crystals.

How can x-ray dye crystals be differentiated from tyrosine crystals?

X-ray dyes greatly increase urine specific gravity to 1.040.

Are tyrosine crystals soluble?

Yes, in alkali or dilute mineral acid.

What gene is mutated in alkaptonuria?

HGD gene.

What enzyme is affected in alkaptonuria?

Homogentisate oxidase.

What pigment changes occur in alkaptonuria?

Dark blue-brown to black pigmentation in cartilage and connective tissue.

What joint condition is associated with alkaptonuria?

Arthritis.

What happens to urine in alkaptonuria?

It darkens or may turn black upon standing or at alkaline pH.

What happens when ferric chloride is added to urine in alkaptonuria?

It turns transient deep blue.

What tests confirm alkaptonuria?

GC and MS/MS.

What enzyme complex is deficient in MSUD?

Branched-chain α-ketoacid dehydrogenase (BCKD).

Which amino acids cannot be normally metabolized in MSUD?

Leucine, isoleucine, and valine.

What odor is characteristic of MSUD?

Maple syrup or burnt sugar smell.

Where can the MSUD odor be detected?

Urine, sweat, and breath.

What severe neonatal findings occur in MSUD? and when does it start?

Vomiting, acidosis, seizures, stupor, failure to thrive, and mental retardation., usually starts when the baby starts drinking milk.

What can happen if MSUD is undiagnosed?

Death.

What inhibitor is used in the modified Guthrie test for MSUD?

4-azaleucine.

What happens in the presence of branched-chain amino acids during the modified Guthrie test?

Bacillus subtilis grows.

What solvent mixture is used in the MSUD microfluorometric assay?

Methanol and acetone.

Why is methanol and acetone added in the MSUD assay?

To denature hemoglobin.

What enzyme is added in the MSUD microfluorometric assay?

Leucine dehydrogenase.

At what wavelength is NADH fluorescence measured in the MSUD assay?

450 nm with excitation at 360 nm.

What leucine concentration is indicative of MSUD?

>4 g/dL.

What prenatal test can diagnose MSUD?

Decarboxylase enzyme concentration in cultured amniotic fluid cells.

What gene is commonly mutated in homocystinuria?

CBS gene.

What enzyme does the CBS gene encode?

Cystathionine β-synthase.

What accumulates in homocystinuria?

Homocysteine and methionine.

What symptoms develop later in childhood in homocystinuria?

Vascular thrombosis, mental retardation, and ocular damage.

What inhibitor is used in the modified Guthrie test for homocystinuria?

L-methionine sulfoximine.

What tests confirm homocystinuria?

HPLC and MS/MS.

What methionine concentration indicates homocystinuria?

>2 mg/dL.

What defect occurs in cystinuria?

Defective reabsorption of cystine and dibasic amino acids by renal tubular cells.

Which dibasic amino acids are affected in cystinuria?

Arginine, lysine, and ornithine.

What develops in cystinuria?

Renal stones.

What finding is diagnostic for cystinuria?

Cystine crystals in urine.

What is cystinosis?

A true inborn error of metabolism.

Where does cystine accumulate in cystinosis?

Cornea, bone marrow, lymph nodes, and internal organs.

What syndrome develops in cystinosis?

Fanconi syndrome with renal failure.

How long should patients fast before blood collection for amino acid analysis?

6–8 hours.

What type of specimen is preferred for amino acid analysis?

Heparinized plasma.

Why must plasma be separated from cells immediately?

To avoid contamination from cells.

How much higher are intracellular aspartic acid and glutamic acid concentrations in WBCs compared to plasma?

100 times higher.

What must be avoided during amino acid analysis specimen collection?

Hemolysis.

When should deproteinization be performed?

Within 30 minutes of sample collection.

At what temperatures should samples be frozen if not immediately analyzed?

-20°C to -40°C.

What type of urine specimen is required for amino acid quantitation?

24-hour urine preserved with thymol or organic solvents.

What role do proteins play in the body?

They play a key role in every cell throughout the body.

What balance is important in protein metabolism?

Balance between catabolism and anabolism.

Nitrogen balance

What is the primary structure of proteins?

The sequence of amino acids genetically encoded by DNA.

What is the secondary structure of proteins?

Recurring coiling formations such as α-helices caused by hydrogen bonding (strength and stability)

What stabilizes tertiary protein structure?

Folded, 3-D conformation of the protein that is stabilized by both covalent bonds and noncovalent forces involving amino acid R groups (function and physical and chemical properties)

What is quaternary protein structure?

Two or more related chains linked by weak chemical bonds (hemoglobin)

What are simple proteins made of?

Amino acids.

How many common amino acids exist?

20

What are examples of conjugated proteins?

Metalloproteins, glycoproteins, phosphoproteins, lipoproteins, and nucleoproteins.

What are examples of catalytic proteins?

Enzymes.

Q: What are examples of regulatory proteins?

Receptors, hormones, repressors, and inhibitors.

What are examples of transport proteins?

Albumin, transferrin, and haptoglobin.

What are examples of protective proteins?

Immunoglobulins and complement.

What is an example of an oncofetal or placental protein?

AFP.

What happens in the Biuret method?

Peptide bonds react with Cu²⁺ ions in alkaline solution to form a purple product.

What does dye binding measure?

Ability of proteins to bind dyes such as Coomassie blue.

What does the turbidimetric method detect?

Protein precipitates/aggregates with SSA or TCA.

What is the order of migration in protein electrophoresis from fastest to slowest?

Albumin, α1 globulin, α2 globulin, beta globulin, gamma globulin.

What is the largest protein fraction on electrophoresis?

A: Albumin.

What is electroendosmosis?

Buffer flow toward the cathode causing the gamma region to become cathodic to the point of application.

What major serum proteins fall into the following peaks:

Albumin:

Alpha1:

Alpha2:

Beta:

Gamma:

Albumin: albumin

Alpha1: alpha1 anti-trypsin and alpha1 acid glycoprotein

Alpha2: alpha3 macroglobulin and haptoglobin

Beta: Hemopexin, Transferrin, beta lipoprotein, and complement C3

Gamma: IgA, IgM, IgG