Kimia Klinis

Mei 2018
 Defisiensi protein merupakan
masalah dunia

Anak umur 2 tahun ini dengan

kwashiokor, sebelum dan sesudah
pengobatan dengan protein yang
sesuai.

Yang mana gambar sebelum dan

sesudah pengobatan?

KWASHIORKOR – defisiensi protein tetapi kalori mencukupi. Thn 1930,

digambarkan sebagai penyakit anak yang mempunyai adik baru.
Karakteristiknya edema.
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 Malnutrisi Protein
Edema pada kelaparan

- Penyebab adalah sintesis protein plasma yang tidak

mencukupi, terutama albumin sehingga tekanan osmotik
tidak dipertahankan dan cairan keluar dari jaringan.
- Cairan tubuh di rongga ekstraseluler meningkat relatif
terhadap bobot tubuh

Cairan ekstraseluler:
Normal ~23.5%
Kwashiokor ~30%

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 Malnutrisi Protein
Selain kelaparan, dapat juga disebabkan oleh:

• kehamilan dan laktasi

• gangguan makan (bulimia, anorexia).
• orang tua dan sakit kronis
• alkoholik kronis dan pengguna narkoba.
• gangguan genetik terhadap absorpsi asam
amino

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▪ Katabolisme atau penguraian protein terjadi
secara kontinu -Dewasa normal : 1-2 % protein
tubuh diganti/hari
▪ Protein diuraikan menjadi asam amino
- 75-80 % → sintesis protein baru
- 20-25 % → amina membentuk ureum
karbon jadi KH dan Lemak
Katabolisme asam amino
Pemecahan asam amino baik berasal dari diet atau
dari biosintesis dimulai dengan melepaskan gugus
alfa-amino dari molekulnya melalui 2 reaksi utama :
SINTESIS UREA
1. Pembentukan Karbamoil fosfat
Disintesis dalam mitokondria dengan enzim sintetase
kabamoil fosfat (irreversible)
Hiperamonemia kongenital tipe I
(kerusakan enzim diatas) timbul gejala :
muntah dan keterlambatan psikomotor dan kadar
glutamin meningkat

2. Pembentukan Sitrulin
Dalam mitokondria dengan enzim transkarbamoilase
ornitin (irreversble)
Kerusakan enzim ini menimbulkan penyakit
Hiperamonemia kongenital tipe II, dengan gejala
seperti diatas dan ditemukannya metabolit pirimidin
dalam urine.
3. Pembentukan Arginosuksinat
Diperlukan enzim sintetase arginosuksinat
- Kerusakan enzim ini menimbulkan sitrulinuria
4. Pembentukan Arginin
Bantuan enzim argino-suksinase
- Kerusakan enzim menimbulkan Arginonosuksinat asidemia
5. Pembentukan Urea
Dibantu oleh enzim arginase
4 5

3 2

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Kelima reaksi diatas terjadi dalam suatu siklus urea dan
kemudian urea dikeluarkan dari tubuh melalui ginjal.
Kegagalan ginjal akan menimbulkan suatu keadaan yang
disebut dengan Uremia
▪ mual, muntah, koma (koma uremik) dan dapat
menimbulkan kematian.
▪ Terapi dengan alat Hemodialisa.
Asam amino yang beredar dalam darah dapat
berasal dari : - katabolisme protein makanan
- sintesis dalam tubuh
Protein makanan
Protein yang berasal dari makanan mengandung 20
macam/jenis asam amino yang penting secara
biologis.
Dalam lambung:
Enzim Pepsin dan Renin dalam suasana asam (pH ±
1) terjadi denaturasi protein.
Dalam sel-sel mukosa usus halus :
- Tripsin dan kimotripsin
- Aminopeptidase dan karboksipeptidase
Asam2 amino yang terbentuk , secara aktif ditransport ke
dalam darah porta dan kemudian ke sel-sel tubuh untuk
homeostasis.
Asam amino yang terus menerus beredar dalam darah 2-
6 mmol/L
Pengeluaran asam amino terutama melalui hati, sedikit
melalui ginjal dan otot.
Sintesis dalam tubuh :
Asam amino hasil pencernaan makanan tidak
terdapat dalam perbandingan seperti yang
diperlukan oleh tubuh, oleh karena itu perlu
disintesis dalam tubuh, terutama asam amino
nonesensial.
Asam amino esensial adalah asam amino yang
tidak dapat disintesis dalam jumlah yang cukup
yaitu : fenilalanin, valin, triptofan, treonin,
isoleusin, metionin, histidin, arginin,leusin dan
lisin (Pvt Tim Hall)
 Alanin, Sistein, Isoleusin,
Glisin, Treonin, Leusin, Lisin,
Triptofan, Serin Treonin

Glukosa Asetoasetat

Asparagin, Leusin, Lisin,

Aspartat Fenilalanin,
Triptofan, Tirosin

Aspartat,
fenilalanin,
Tirosin

Isoleusin, Arginin, Glutamat,

Metionin, Valin Glutamin, Histidin,
Prolin
 KELAINAN BAWAAN METABOLISME ASAM AMINO
1. Fenilketonuria
- Defisiensi enzim fenilalanin hidroksilase
- Cacat bawaan
- Keterlambatan mental
- Dalam urine terdapat
* fenil piruvat
* fenil asetat
* fenil laktat
2. Alkaptonuria
- Defisiensi enzim homogentisat oksidase
- Homogentisat hitam
- Klinis tak ada
3. Sistinuria
- Kelainan ginjal / absorbsi kurang
- Batu ginjal (sistin)
- Dalam urin terdapat : * sistin
* lisin
* arginin
 Proteins: Common properties
• In spite of functional differences between the various
serum proteins, they have certain common
biophysical and biochemical properties. These
include:
– a basic composition of carbon, hydrogen, nitrogen
and oxygen;
– a backbone of covalent peptide bonds which join
the amino acid units together; and
– absorption maxima in the ultraviolet region.
• Based on these properties, laboratory methods have
been developed to determine the concentration of
proteins in serum,
Mohammed Laqqan
 Serum Total Protein
• Serum total protein, also called plasma total protein or total
protein, is a biochemical test for measuring the total amount of
protein in blood plasma or serum.
• Protein in the plasma is made up of albumin and globulins.

• Note: the globulin in turn is made up of α1, α2, β, and γ

globulins.

• These fractions can be quantitated using protein

electrophoresis, but the total protein test is a faster and cheaper
test that estimates the total of all fractions together.
• The traditional method for measuring total protein uses the
biuret reagent, but other chemical methods are also available.

Mohammed Laqqan
 Methods of Total Protein Analysis

• Method 1: Kjeldahl; quantitative, protein nitrogen

determination

• Method 2: Biuret; quantitative, increased absorption

at 540 nm;

Mohammed Laqqan
 Specimen
• Serum and plasma may be used, and all usually
yield comparable results, though, because of the
presence of fibrinogen, plasma levels for total
protein are 2 to 4 g/L higher than serum levels.
• A fasting specimen is not required but may be
desirable to decrease lipemia.
• Total protein is stable in serum and plasma for
– 1 week at room temperature,
– and for at least 2 months at –20° C

Mohammed Laqqan
➢ Hypoproteinemia
– Malnutrition and/or malabsorption
– Excessive loss as in renal disease, GI leakage,
– excessive bleeding, severe burns
– Excessive catabolism
– Liver disease

➢ Hyperproteinemia
– Dehydration
– Monoclonal increases
– Polyclonal increase

• Only disorders affecting the concentration of albumin and/or the

immunoglobulins will give rise to abnormal total protein levels.
• Other serum proteins are never present in high enough concentrations for
changes to have a significant overall effect.

Mohammed Laqqan
 The Biuret Method
• The Biuret reagent is made of (NaOH) and copper (II) sulfate
(CuSO4), together with potassium sodium tartrate
(KNaC4H4O6).
– A blue reagent which turns violet in the presence of
proteins.
• The Sodium hydroxide does not participate in the reaction at
all, but is merely there to provide an alkaline medium so that
the reaction can take place.

Mohammed Laqqan
 Principle: Biuret Method
• Peptide bonds of proteins react with tartrate-
complexed cupric ions in alkaline solutions to form a
colored product.
• In a positive test, a copper(II) ion is reduced to
copper(I), which forms a complex with the nitrogens
and carbons of the peptide bonds in an alkaline
solution.
• A violet color indicates the presence of proteins.
• The intensity of the color, and hence the absorption at
540 nm, is directly proportional to the protein
concentration, and can be determined
spectrophotometrically at 540 nm.
Mohammed Laqqan
 Reference range

• Reference range for total proteins is 66.6 to 81.4 g/L

• Results for males are approximately 1 g/L higher than results
for females; this difference is probably not of clinical
significance.
• In newborns, the mean serum protein concentration is 57 g/L,
increasing to 60 g/L by 6 months and to adult levels by about 3
years of age.
• Serum protein levels of premature infants can be much lower
than that of full term infants, ranging from 36 to 60 g/L.

Mohammed Laqqan
 Albumin
• Albumin is the most abundant circulating plasma
protein (40–60 % of the total)
• Playing important roles in the maintenance of the
colloid osmotic pressure of the blood, in transport of
various ions, acids, and hormones.
• It is a globular protein with a molecular weight of
approximately 66,000 D and is unique among major
plasma proteins in containing no carbohydrate.
• It has a relatively low content of tryptophan and is an
anion at pH 7.4.

Mohammed Laqqan
 Analysis Methods
• Method 1: Precipitation; quantitative
– Salt fractionation, Acid fractionation
– Principle of analysis: Changes of net charge of protein
result in precipitation
• Method 2: Tryptophan content; quantitative
– Principle of analysis:
– Glyoxylic acid + tryptophan in globulin Purple chromogen
(Amax, 540 nm); Total protein – globulin = albumin.
• Method 3: Electrophoresis; quantitative
– Principle of analysis: Albumin is separated from other
proteins in electrical field; percent staining of albumin
fraction multiplied by total protein value

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• Method 4: Dye binding, quantitative
– Methyl orange; BCG (bromcresol green); BCP (bromcresol
purple);

• Method 5: Dye binding; semiquantitative

– Bromphenol blue in test strip changes color from yellow to
blue in presence of albumin most commonly used test for
urine protein

Mohammed Laqqan
• Specimen: Serum is the specimen of choice, but heparinized
plasma can also be used if precautions are taken to prevent
heparin interferences.

• Interfering Factors
– Albumin is decreased in:
• Pregnancy (last trimester, owing to increased plasma
volume)
• Oral birth control (estrogens) and other drugs.
• Prolonged bed rest.
• IV fluids, rapid hydration, overhydration.

Albumin Reference Interval for Serum

Age Men (g/L) Women (g/L)
21–44 33.3–61.2 27.8–56.5
 Clinical Significance
• Plasma albumin levels, although important for management
and follow-up, have very little value in clinical diagnosis.
• Hyperalbuminemia is usually attributable to
• dehydration or hemoconcentration.
• Hypoalbuminemia is usually the result of
• hemodilution,
• a rate of synthesis less than the albumin loss,
• diseases that cause a large albumin loss from urine,
skin, or intestine,
• increased catabolism observed in fevers, untreated
diabetes mellitus, and hyperthyroidism.

Mohammed Laqqan
 Dye-binding Techniques

• Serum albumin is most often assayed using dye-binding

techniques.
• Albumin preferentially binds to anionic dyes that do not attract
globulins
• Bromcresol purple (BCP) and bromcresol green (BCG) are
most commonly used
• The amount of light absorbed by the albumin –dye complex is
proportional to the amount of albumin present

Mohammed Laqqan