CARBOHYDRATES The net effect is to return blood glucose level to normal.

One function is to
Aldehyde or ketone derivatives of polyhydric alcohols increase the uptake of glucose from the blood to the target cell. If no insulin,
primary source of energy for the body together with lipids there is no means to mediate the glucose into the cells.
Atwater factor = 4 kcal
If one does not eat meal, hypoglycemia occurs (60 mg %). This stimulates the
Glucose: Glucose + ATP -Hexokinase-> Glucose -6-PO4 + ADP alpha cells of pancreatic enzymes of Langerhans cells and release glucagon.
This accelerates glycogenolysis, Gluconeogenesis. The effect is to elevate the
Oxidative Pathways blood glucose level.
1. Glycolysis
2. Krebs Cycle Hormones Controlling Blood Glucose Concentration
3. Hexose Monophosphate Shunt - Pentose Phosphate Pathway Hypoglycemic Hormones:
Source of NADPH 1. Insulin - primary hypoglycemic factor; secreted by the beta-cells of the
Result of reducing equivalence in the form NADPH pancreatic islets of Langerhans
Maintain membrane protein in the reduced phase to withstand Stimulates glycogenesis
reactions Enhances transport of glucose from blood to target cells
Source of pentoses (ribose for synthesis of DNA and RNA) Stimulates lipogenesis
Generated CO2 2. Somatostatin - secondary hypoglycemic factor; secreted by the delta cells of
4. Uronic Acid Pathway the pancreatic islets
An oxidative pathway that does not generate ATP, but generates carbon Inhibits secretion of glucagon by the alpha-cells o the pancreatic
dioxide and pentoses, and a source of ascorbic acid which is essential to islets
the human body
Used by liver in conjugating toxic substances Hyperglycemic Hormones:
1. Glucagon - primary hyperglycemic factor; secreted by the alpha-cells of the
Synthetic Pathways pancreatic islets of Langerhans
1. Glycogenesis - formation of glycogen from glucose units Stimulates hepatic glycogenolysis
Occurs in liver and muscles Diabetes Milletus - caused by virus which targets beta cells of pancreas
2. Lipogenesis - connected to glycolytic pathway 2. Epinephrine (adrenalin) - secreted in "stressful situations" by the adrenal
Uses excess product of glycolytic pathway medulla
3. Gluconeogenesis - formation or synthesis of glucose from non-carbohydrate Stimulates glycogenolysis
sources Stimulates lipolysis
3. 11-Oxysteroid - secreted by the adrenal cortex
Other Catabolic Pathway Stimulates gluconeogenesis
1. Glycogenolysis - breakdown of glycogen to produce glucose units 4. Adrenocorticotropic hormone (ACTH) and Growth Hormone - secreted by the
anterior pituitary gland
Normal blood glucose level: ACTH - stimulates adrenal cortex to secrete the steroids, e.g. 11-
70 - 100 mg % oxysteroids; indirectly increase blood sugar
3.5 - 5.5 mmol/L Growth hormone - stimulates gluconeogenesis
5. Thyroxine - secreted by the thyroid gland
Blood glucose rises after eating. Stimulates intestinal absorption of glucose
Presence of hyperglycemia (120 mg %)stimulates the beta cells of pancreatic Stimulates glycogenolysis
enzymes of Langerhans cells and release insulin. This glycolysis, TCA,
Alt. oxidative pathway, Glycogenesis, Lipogenesis Specimen Used in Glucose Determination
1. Whole blood:
Blood glucose value varies with the hematocrit
 Glucose in whole blood is unstable Principle Involved: a two-step enzymatic reaction
7mg glucose/100 mL/hr = if no preservative is added; due to 1. Glucose + ATP -(Hexokinase)-> Glucose-6-PO4 + ADP
oxidation 2. Glucose-6-PO4 + NADP+ -(G-6PO)-> 6-
Cannot be used in automated methods for glucose Phosphogluconolactone + NADPH (340nm)
If it inhibits enolase, glucose level is preserved G-6-PD-Glucose-6-PO4 dehydrogenase
In automated machines, specimens are separated by a bubble and The amount of NADPH formed is to the amount of glucose
passes through different parts of the machine present in the sample
2. Serum
Preferred specimen because it has no red cells (we allow blood to clot) b. Colorimetric method:
Glucose is stable within 8 hours at room temperature and 24 hours if Uses a chromogenic system that will produce a colored end
refrigerated product
3. Plasma Principle involved: Utilizes steps 1 and 2 of the non-colorimetric
Glucose is stable if the specimen is not contaminated with red cells method
o NADPH + PMS-INT -> NADP+ + reduced chromogen
Methods of Blood Glucose Determination (520 nm)
General Methods: o PMS - Phenazine methosulfate
I. Enzymatic Methods (uses enzymes) o INT - Iodophenyl nitrophenyl tetrazolium chloride
1. Hexokinase Method The amount of light absorbed by the reduced chromogen
a. Non-colorimetric - the product produced is not colored; cannot (colored) is to the amount of NADPH which in turn is to the
use photometry amount of glucose in sample
b. Colorimetric
2. Glucose oxidase method 2. Glucose oxidase method
a. Colorimetric
Glucose + O2 + H2O - -> Gluconic acid +H2O2
b. Polarographic
c. Automated
II. Chemical Methods a. Colorimetric method
A. Copper Reduction Methods Principle involved: Based on a two-step reaction:

1. Folin-Wu Method 1. Glucose + O2 + H2O - -> Gluconic acid +H2O2

2. Somogyi-Nelson method
2. H2O2 + Chromogenic oxygen acceptor -(peroxidase)->
3. Benedict's method H2O + Oxidized COA
4. Neocupreine method
COA - may be the ortho-dianisidine; its oxidized form may be
B. Ferricyanide Reduction Method
yellow or pink in color depending on the pH
1. Automated method
o Yellow (slightly acidic) - measured at 395 nm
C. Condensation Method
o Pink (highly acidic) - measured at 540 nm (in most
1. Ortho-toluidine method (Dubowski's method)
commercial kits)
2. Condensation with other aromatic amines,e.g. aniline, biphenyl
Disadvantage of the method:
o Interfering substances will compete with COA
Enzymatic Methods
producing a falsely result for glucose
1. Hexokinase method:
o Ex. Uric acid, ascorbic acid, bilirubin, glutathione,
Reference method for blood glucose determination; most accurate and
creatinine
precise
b. Polarographic method
a. Non-colorimetric method:
Final solution is not colored
 Uses only the glucose oxidase reaction and not the Constriction - prevents preoxidate to precipitate (contains Cu2O or
peroxidase reaction Cu(OH)2)
Does not measure the absorbance but the rate of utilization Principle Involved: Involves a two-step reaction
of oxygen 1. Glucose is oxidized to gluconic acid and cupric ions are reduced to
The more glucose is present in the sample, the greater is the cuprous ions
rate of utilization of O2
Glucose + Cu++ - > Gluconic acid + Cu+
Disadvantage of the method:

o The presence of contaminating enzyme, catalase, Green, yellow, orange, brown, to brick red colored
which decompose H2O2 to O2, thereby interfering in precipitate after the first step.
the accuracy of the measurement 2. Cu+ is oxidized back to Cu++ and phosphomolybdic acid is
reduced to phophomolybdus acid (blue)
3. Automated method Cu+ + Phosphomolybdic acid -> Cu++ + Phosphomolybdous
acid (blue-520 nm)
Utilizes the principle of the colorimetric glucose oxidase and peroxidase
reactions Added with water and the tube is inverted.
The absorbance of the blue color produced is to the amount of
1. Glucose + O2 + H2O -( ) -> Gluconic acid +H2O2 Cu+ present which in turn is to the amount of glucose in the

2. H2O2 + Methyl benzothiozolinone hydrazone + Dimethylaniline - sample

-> Indamine dye (600nm) + H2O + OH- Normal value (N.V.) = 80-120 mg %
o Higher because saccharoids are not removed
Absorbance is to the indamine dye produced in the reaction which in
N.V. = 70 - 100 mg %
turn is to the amount of glucose in the sample
o Saccharoids are removed
Trinder Modification of Glucose Oxidase Method
2. Somogyi-Nelson method:
An alternative automated method
The modification is in step no. 2 which is replaced by the following: Removes saccharoids
Zinc hydroxide PFF(S-N method)
H2O2 + Phenol + Para-amino pherazone (PAP) - -> Colored product Reagents: cupric hydroxide & arsenomolybdic acid
(565 nm) + H2O Principle Involved: Utilizes a two-step reaction
1. Same as in Folin-Wu
The above principle is used in the following commercial kits: 2. Cu+ + arseonmolybdic acid -> Cu++ + Arsenomolybdous acid (blue
a. GOD-PAP - 520 nnm)
b. Glucofix
3. Benedict's method:
Chemical Methods Less commonly used
A. Copper Reduction Methods Routinely used qualitative sugar determination
1. Folin-Wu method:
Folin-Wu or Tungstic acid PFF (protein-free filtrate) 4. Neocupreine method:
Reagents: cupric hydroxide & phosphomolybdic acid Utilized in other automated machines
Folin-Wu tube Principle Involved: Based also on a two-step reaction
1. Same as the copper reduction methods
2. Not a redox reaction but a condensation reaction
Cu+ + 2,9-Dimethyl, 1, 10 phenanthroline (neocupreine
reagent)-> orange-colored complex
The absorbance of the orange-complex is to the amount of
glucose in the sample
 Using ortho-toluidine method: range is 4 % higher
B. Ferricyanide Reduction Method Using Folin-Wu method: 80-120 mg% or 4.4 - 6.6 mmol/L
Utilized also in other automated machines Using Somoygi-Nelson method: 70-100 mg% or 3.85 - 5.5. mmol/L
Principle Involved: Based on inverse colorimetry
o Ferricyanide ion (yellow) -(glucose)-> ferrocyanide ion (colorless) Classification of Hyperglycemia
measured at 420 nm Types of Diabetes Mellitus
The decrease in absorbance will be to the amount of glucose in the I. Insulin - Dependent Diabetes Mellitus (IDMM)
sample. Synonyms: Type I Diabetes, Type 1 Diabetes, Juvenile-onset Diabetes

C. Condensation Methods Features:

Qualitative tests for sugars in Biochemistry, Severe/overt diabetes
Anthrone test, Molisch test Not-age specific but common in younger age groups
There is absolute deficiency in insulin due to destruction of the beta-cells
In clinical chemistry, cannot be used in automated machines of the pancreatic islets of Langerhans (e.g. viral infection, autoimmune
1. Ortho-toluidine method (Dubowski's method) antibodies)
Uses ortho-toluidine, an aromatic compound Characterized by ketosis
Principle Involved: Acidosis
O-toluidine + Glucose -
+
-> Glucosylamine - Ketonemia - presence of ketone bodies in the blood
Ketonuria - presence of ketone bodies in the urine
Dehydration
Hyperglycemia and glycosuria
-> Schiff's base (green color) measured at 630 nm Rapid and deep breathing (response of the lungs to acidosis in order to
restore the pH)
The intensity (abs) of the green color is to the amount of glucose Coma - acidosis depresses the functioning of the central nervous system
in the sample
2. Other condensation methods using: Signs and Symptoms
Aniline Hyperglycemia - absolute deficiency of insulin
Biphenyl Polydipsia - excessive thirst, osmotic diuresis (fluids)
Polyuria - frequent urination of large volume of urine specific
Basic Consideration in Selection of Method gravity
1. Method must use stable, harmless reagent Diabetes insipidus - urination of large volume of urine specific
2. Procedure should be simple, rapid, sensitive, precise, accurate and yet gravity
relatively cheap Deficiency in ADH secretion
Ketosis
Normal/Reference Values for Blood Glucose Acidosis - blood pH
SI Unit = mmol/L Ketonemia
Conventional uit = mg % Ketonuria
Conversion factor = 0.055; mg % (0.055) = 0.055 glucose conc. in mmol/L Angiopathy
Myopathy
Adult Values: Retinopathy
Using enzymatic method: Lens of the eye cataract
Specimen = Plasma or serum: 60-110mg% or 3.3 - 6.1 mmol/L) Nephropathy
Specimen= whole blood: 15% lower) Neuropathy
Specimen = PFF from serum/plasma: 5% higher) Hemangiopathy
 Renal threshold - 160-180 mg % (if higher, excess will be deposited into o Disadvantage:
the urine) Amount and duration of meal can't be controlled
3. Random Blood Sugar (RBS)
II. Non-insulin Dependent Diabetes Mellitus 4. Post Challenge Glucose Determination
Synonyms: Type II Diabetes, Type 2 Diabetes, Adult-onset Diabetes Mellitus, o Similar to PPBS but patient is given a standard meal; also a screening test
Mild Diabetes, Chemical Diabetes Glucose solution
100 g glucose/200 mL solution
Subtypes: May be flavored with calamansi juice
1. Associated with obesity Uses cold water as a solvent
2. Not associated with obesity Commercially available cola-flavored glucose solution
Dextrosol
Features: Canned pure dextrose solvent
Due to defective secretion of insulin, deficiency of insulin and resistance 100 g 100mL solution + calamansi
of target cells/organ to insulin Glucola
Ketosis - resistant type of diabetes 100 g for adults
Can be managed by diet and exercise 0.75 g/kg BW for children and juveniles
III. Gestational Diabetes mellitus o Interpretation:
Synonym: Latent Diabetes mellitus Glucose value
<140 mg% not diabetic
Features: >200 mg% suggestive of DM
Seen in some women during pregnancy Has to be confirmed by other tests
May return to normal after delivery 5. Oral Gluose Tolerance Test (OGTT)
May progress to adult-onset DM o Standard test to diagnose mild diabetes (110 mg %)
o Also used to detect hyperinsulinism (consistent hypoglycemia)
IV. Others o Procedure:
A. Preparation Prior to the Test
Diagnostic Strategies for Diabetes Mellitus Eat meals with at least 150g or more CHOs three days prior
1. FBS Determination: to the test
a. Type I Unlimited physical activity
Characterized by fasting hyperglycemia Abstain from medication at least three days before the test
FBS > 140 mg% (7.7 mmol/L) after at least 2 meals diagnostic of Fast for 10-16 hours one a day before the test
DM B. Test Proper
b. Type II Should not smoke, eat anything nor indulge in physical
FBS may still be normal. e.g. 5.3 mmol/L (high normal) can't be activity
diagnosed simply by FBS Fasting blood sample and urine sample are collected and
Other tests should be used refrigerated
2. Post-Prandial Blood Sugar (PBBS) Determination Glucose meal is given orally (200 mL) to be consumed
o Only a screening test for DM within 5-10 mins
o The patient eats a regular meal (no fasting); 2 hours after blood is After meal, blood is collected after 30 mins, 1 hr, 2 hrs, 3
extracted and determined for glucose hrs (urine samplesare similarly collected at the same time
o Interpretation: intervals)
Glucose concentration < 140 mg% (7.7 mmol/L) excludes DM Test is extended to 4-5 hrs if purpose is to detect
Glucose concentration > 200 mg% (11.0 mmol/L) is dagnostic of hyperinsulinism
DM and has to be confirmed by other tests
 Blood on urine samples are then analyzed for glucose 3. In Type 2 (mild) DM
concentration at the same time Time FBS Result Urine Glucose
C. Interpretation of Results
Based on the principle that a normal individual is able to 0 1200 mg % -
dispose of excess glucose 2-3 hrs after a meal
1. In a normal person 30 mins 210 mg % +
Time FBS Result Urine Glucose 1 hr 180 mg % T (trace amount)
0 90 mg % - 2 hrs 160 mg % -
30 mins 160 mg % - 3 hrs 145 mg % -
1 hr 150 mg % - Features:
a. Fasting glucose may be normal or slightly
2 hrs 110 mg % - increased
b. Peak glucose value is higher than normal renal
3 hrs 90 mg % - threshold for glucose
Features of a Normal OGTT c. 3hrs after a meal, the glucose value is still high
a. Absence of fasting hyperglycemia compared to the fasting level
b. Blood glucose level peak after 30min - 1hr Patient has low glucose tolerance
after the standard meal (delayed metabolism of glucose)
c. The peak glucose value d. One or more of the urine specimen is (+)

2. In Type I Diabetes Mellitus 4. In Hyperinsilunism - syncope, insulinoma (tumor

secreting insulin)
Time FBS Result Urine Glucose
Time FBS Result Urine Glucose
0 210 mg % +
0 50 mg % -
30 mins 260 mg % +
30 mins 70 mg % -
1 hr 320 mg % ++
1 hr 60 mg % -
2 hrs 370 mg % +++ brown
2 hrs 55 mg % -
3 hrs 430 mg % ++++ orange to brick
red 3 hrs 45 mg % -
Features: Features:
a. Fasting hyperglycemia a. Fasting hypoglycemia
b. Peaking of blood glucose concentration is b. After 30mins, blood glucose is not significantly
gradual increased
c. Blood glucose level may reach as high as >460 c. 3 hrs after a meal, value is still lower than FBS
mg% D. Criteria Used in Interpreting OGTT Results
d. After 3hrs, blood glucose value is still very 1. Wilkerson Point System
much elevated 2. University Group Diabetes Program
e. After 4hrs, value may start to decline 3. Fajan-Conn Criteria
 6. Intravenous Glucose Tolerance Test (IGTT) Glucose is attached to N-terminal valine of Hb chain
o Differs from OGTT in the manner of administration HbA1c concentration is directly proportional to the
o Done when patient has malabsorption syndrome, intestinal surgery, etc time-averaged blood glucose concentration for the
o 25% glucose solution is used at a dosage of 0.5g/kg BW past 2-3 months prior to the test
o Prior to IV glucose administration, FBS determination is done after which HbA1c concentration is not affected by sudden
blood glucose is determined every 10 min for one hour (total of 7 fluctuation in blood glucose level
venipunctures) o Methods of Glycosylated Hb Determination
o Test duration is shorter than OGTT 1. Ion-exchange Chromatography
o Results: 2. High-performance Liquid Chromatography
10 min 190 mg % 40 min 120 mg % 3. Electrophoresis
4. Radioimmunoassay
20 min 170 mg % 50 min 95 mg % 5. Colorimetric Spectrophotometry - cheapest

30 min 140 mg % 60 min 70 mg %

o Compute for Kt 1/2
Time in minutes required for blood glucose value to decrease by 1/2
the 10 minimum values after glucose administration
70 70
Constant = = x 100% = 1.4
50
o Interpretation:
1.5% - normal
<1.5% - abnormal or diabetic

Monitoring & Control of Diabetes Mellitus

Tests used:
1. FBS and Urine Sugar Determinations
o Done regularly at intervals
2. Home monitoring/Self-monitoring
o Uses rapid strip tests - dip the strip and compare it to a color block
a. Glucostix o Normal values for HbA1:
b. Dextrostix 6.5-8.0% of total Hb
3. Measurement of Glycosylated Hemoglobin
8.0-14.0% in diabetic patient
o Glycosylated hemoglobin - Hbs with attached carbohydrates, also known
>14% = poor control of DM
as "glycated" or "fast" Hb
o "Fast" Hb - travel faster than non-glycosylated Hb on chromatography
PROTEINS
and electrophoresis
From the word "proteus" which means most important or the first
o In normal adults:
Found in the plasma (peripheral and integral)
HbA = 95-98% (includes glycosylated Hb) Function as antibodies and immunoglobulins
HbA2 = 2-3% For movement (in muscles, actin, myosin); contractile proteins
HbF = about 1% For transport (albumin, transferrin(iron))
Glycosylated Hbs (HbA1) Regulation of chemical processes as peptides and hormones
HbA1a = contains fructose
Fluid volume regulation (albumin)
HbA1b = contains glucose-PO4
Osmotic regulation (albumin)
HbA1c = contains glucose (most important)
 Interferon
(CRP) inflammations
R C skeleton ---->glucose
-----> fatty acid Gamma () - Globulins Are Antibodies:

Proteins----------->H-C-NH3 ------------------->NH4 > UREA Immunoglobulin G * in late response
()

Immunoglobulin A * in secretion
COO >-----> CO2 IgD * in tissue reaction
IgM in early response
Amino Acid pool IgE reagins; allergic rxn

Selected Plasma Protein
PROTEIN
Prealbumin (Transthyretin)
TPAG - total protein albumin globulin; total protein in the plasma
COMMENTS
Fractionation of Plasma Proteins by Electrophoresis
Indicator of nutrition; binds thyroid hormones and
retinal binding protein (RBP)

Albumin Binds bilirubin, steroids, fatty acids (Alb-FFA);

maintenance of blood osmotic pressure fluid volume
regulation

1 Globulins
1 - Antitrypsin Acute phase reactant; protease inhibitor
1 - Fetoprotein Principal Fetal protein
1 - Lipoprotein (HDL) Transports cholesterol and other lipids
Methods of Protein Determination
1 - Antichymotrypsiin
I. Methods for Total Protein
Gc Globulin Inhibits chymotrypsin
1. Total Nitrogen Determination
e.g. Kjeldahl method
Binds Vit. D and actin
2. Refractometry
2 Globulins 3. Biuret
Haptoglobins Acute phase reactant; binds Ab 4. Dye-binding
Ceruloplasmin Peroxidase activity; contains Cu++ 5. Ultraviolet Absorption
2 - Macroglobulin Inhibits thrombin, trypsin, pepsin II. Methods for Albumin
1. Salt Precipitation
Globulins 2. Dye-binding
Pre - Lipoproteins Transports triglycerides e.g. Methyl orange
(vLDL) HABA [2(4'-hydrocyazobenzene)-benzoic acid]
Transferrin Transports iron BCG (bromcresol green)
Hemopexin Binds heme BCP (bromcresol purple)
- Lipoprotein (LDL) Transports cholesterol 3. Electrophoresis
- 2 Microglobulin Component of human leukocyte antigen (HLA) III. Methods for Urine Proteins
(B2M) 1. Turbidimetric Methods(sulfosalicylic acid, TCA or benzethonium chloride
Complement Immune response 2. Biuret
Fibrinogen Precursor of fibrin clot 3. Folin-Lowry
C-reactive Protein Acute phase reactant; motivates phagocytosis in 4. Dye-binding
 e.g. Coomassie blue, Ponceau S o Results are quite satisfactory
o Most practical
A. Methods for Total Protein Principle involved:
1. Total Nitrogen Determination (Kjeldahl Method) Based on the formation of a violet or purple-colored chelate or
Classical method for quantitation of total proteins coordination complex between Cu ++ and peptide bonds of
Reference method because of its accuracy and precision proteins. The absorbance of the solution is to the protein conc.
Assumes N2 content of proteins of 16% In the sample (feeling ko kulang)
Not routinely used
Principle Involved: 4. Dye-binding method:
Proteins in the sample are digested and the total nitrogen content e.g. Method of Bradford
measured Uses the Coomasie blue 250 dye
Steps in Procedure: Principle Involved:
a. Proteins in the sample are precipitated with tungstic acid The dye (Coomassie blue 250) binds with proteins in the serum
b. Wash protein precipitate sample causing a shift in the absorbance maximum of the dye
c. Oxidation (digestion) of ppt. in a Kjeldahl flask to release NH4+ from 465 nm to 595 nm. The increase in absorbance at 595 nm is
d. Measurement of the amount of NH4+ liberated by: used to determine the protein concentration.
i. Nesslerization
NH4+ + Nessler's rgt. (Mercuric potassium iodide or Double 5. Ultraviolet Absorption:
iodide) dimercuric ammonium iodide (yellow) Based on the ability of proteins to absorb light at 210 nm and 280
spectrophotometric measurement nm. This is due to their content of aromatic amino acids - TYR, TR
ii. Titration with an Acid & PHE
e. Computation of protein concentration using the following:
Protein conc. = N x 6.25 (g/L) B. Methods for Albumin
1. Salt Precipitation/Fractionation:
2. Refractometry Based on the fractionation of proteins by precipitation
Rapid and simple method for both serum and urine total proteins Can be used to determine "TP", "A", "G", and A/G ratio (TP, AG)
Assumes nonprotein solids are present in the same conc. as in
the calibrating serum Procedure:
Majority of solids in serum are proteins, therefore, the refractive a. "TP" determination
index reflects protein conc. Diluted serum sample + Biuret reagent (alkaline copper
Has a reported agreement of + 3% with Biuret sulfate)
Principle Involved: Violet color
This is based on the measurement of refractive index (R.I) due to b. Add 23% sodium sulfate or 24% sodium sulfate
the presence of solutes in the serum sample
C. "A" determination
3. Biuret Reaction: Shaken vigorously and centrifuged with other
Specific methods: Globulins pptd as "cake"
Kingsley (author of one Biuret method) Albumin will be found in the bottom layer; analyzed for the Biuret rxn
Weichelbaum
Reinhold
Basic Reagent: CuSO4 and NaOH
Most widely used method and the one recommended by the IFCC
because:
o Easy to perform
 G = 1.5 - 3.5 g% or 15 - 35 g/L
A/G ratio: 1.3 :1

1. Dye-binding
Most widely used method for albumin
pH of solution adjusted to make albumin (+) charged and reactive to
anionic dyes
e.g. HABA, BCG, & BCP

2. Electrophoresis

Other Methods for Protein Determination

1. Folin-Ciocalteau Method
Uses phosphotungstomolybdic acid (oxidizing agent)
Can be used to detect proteins with
o TYR (phenol ring)
o TRP (indole ring)
o HIS (imidazole ring)
o All of the above has weak reducing agents

Principle Involved:
D. Compute for "TP", "G", "A", and A:G ratio Proteins containing Tyr, Trp, & His can be oxidized by
TP & A = from standard calibration curve phosphotungstomolybdic acid to a colored complex. The
G = TP-A intensity/absorbance of the colored complex is directly proportional to
A/G ratio: expresses as x : 1 the concentration of Tyr, Trp, % His in the solution which in turn is
directly proportional to protein conc.
Ex. Result of TP, AG determination 5-6x more sensitive than Biuret
TP = 7.5 g/100mL Has the disadvantage that different proteins have varying
A = 8.0 g/100 mL amounts of Ty, Trp, & His
G = 5.5 g/100 mL
2. Lowry Method
Combination of Biuret & Folin-Ciocalteau methods
1. Cu = Used routinely for CSF protein determination

2. Standard calibration curve
Conventional: g/100 mL; SI : g/1000mL or 1L C. Methods for Urine Protein
A/G ratio: 1. Turbidimetric Methods
A:G = x:1 Uses sulfosalicylic acid, TCA & benzethonium chloride
2.0 : 5.3 = x:1 Rapid; easy to use; unequal sensitivity for individual proteins
X = 0.3 Principle Involved:
A/G ratio = 0.36 : 1 o Proteins are precipitated as fine particles & turbidity is
measured spectrophotomtetrically
Normal values: 2. Biuret
TP = 6.0 - 8.0 g% or 60 - 80 g/L Accurate
A = 3.5 - 5.5 g% or 35 - 55 g/L 3. Folin-Lowry
 Very sensitive 3. Creatine
4. Dye-binding (Coomassie blue, PonceauS) 4. Uric Acid
Limited linearity; unequal sensitivity for individual proteins 5. Amine acids
6. Nucleotides
Methods of Protein Fractionation: 7. Polypeptides
Separation of different types of proteins in a given sample 8. Glutathione
1. Salt Fractionation Methods 9. Others
2. Ultracentrifugation o The first four are the most clinically important
3. Chromatography
4. Electrophoresis Total NPN: Formerly requested for diagnosis of renal impairment but not anymore
today
Ultracentrifugation Urea & Creatinine determination = routinely requested now in place of NPN
Separate proteins on the basis of varying densities
Uses NaCl of known specific gravity and high speed centrifugation UREA
Used in separating the plasma lipoproteins Major excretion product of protein catabolism
Chylomicrons <0.96 Synthesized in the liver via the urea cycle (ureagenesis)
Methodology:
vLDL 0.96 - 1.006 1. Indirect method
2. Direct method
LDL 1.006 - 1.063
Indirect Method:
HDL 1.063 - 1.210
Involves 2 steps
1. Urease reaction
Clinical Application of Protein Determinations
A. Conditions Associated with Protein Concentration:
1. Dehydration - vomiting, diarrhea, etc.
Solvent decreases
2. Multiple myeloma - plasma cells produces too much plasma proteins
3. Other conditions, e.g. neoplasm, chronic inflammatory condition

B. Conditions with Protein Concentration (Hypoproteinemia): Amount of NH4+ released is to the amount of urea in the
1. Renal disease (e.g. nephrotic syndrome) sample (BUN Level is obtained)
2. Inflammation of GIT BUN - Blood urea nitrogen
Gastric cells become necrotic; cells are made of proteins; losing 2. Measurement of the amount of ammonia released from urea using one
large amount of gastric cells loses proteins of the following:
3. Loss of blood a. Berthelot reaction
b. Glutamate dehydrogenase reaction
NON-PROTEIN NITROGEN (NPN) SUBSTANCES c. Amount of electrical conductivity
Heterogeneous group of substances most of which are end-product of d. Potentionmetry
metabolism
a. Berthelot reaction
Includes: Uses the following:
1. Urea Sodium hypochlorite (directly involved in the reaction)
2. Creatinine
 Phenol (directly involved in the reaction)
Sodium nitroprusside - catalyst
Principle Involved:
o NH4+ + 5 sodium hypochlorite + 2 Phenol Idophenol
(blue) + 5 NaCl + 5 H2O
o Absorbance of the blue color is directly proportional to
the amount of indophenol formed which in turn is too
the concentration of NH4+ in turn is to the concentration
of urea present in the sample.
URASTAT
A commercially-available strip test for indirect determination of urea
A chromatographic strip test impregnated with 3 reagent zones:
1. Urease zone - near one end
2. K+ carbonate zone
3. Indicator zone - bromcresol green in tartaric acid

b. Glutamate dehydrogenase reaction

Basis for many automated of BUN determination
Principle Involved:
Immersed in a test tube with 0.02 mL serum
o Glutamate + NADP+ H2O > -ketoglutarate + NADPH Allowed to stand for 30 min.
+ H+ When serum reaches indicator zone, indicator changes in color
o Quantitation involves use of reverse reaction. Decrease o Yellow green or blue-green
in absorbance at 340 nm is monitored. The rate of Height of the green color (mm) measured
change in A at 340 nm is amount of NH4+ release from o Height (mm) BUN concentration
the reaction symbol amount of urea originally present in Expensive; not used routinely
the specimen
Direct Method
c. Conductimetric Method Methods based on the REARON reaction
Principle Involved: Diacetyl + Urea Diazine Derivative + 2H2O
o Based on the release of NH4+ and CO3- from urea by the Diacetyl monoxime = reagent used; source of diacetyl
action of urease on the specimen placed in a Diazine derivative formed is measured at 540 nm
conductimetric cell. The in the conductivity of the A concentration of urea in sample
solution is measured and is directly proportional to the
amount of urea in the sample. 1. Friedman Method
o Conversion of Urea Concentration to BUN:
d. Potentiometric Method Urea concentration = 40 mg%
Rarely applied BUN = 40 mg% x 28/60
Uses a special NH4+ sensing electrode which can detect presence 18.8 mg%
of NH4+ released by the action of urease on the urea present in o Normal values:
the sample. BUN:
Adults = 7-18 mg%
Conversion of BUN to Urea Concentration Neonates = 4-12 mg%
Conversion factor = 2.14 Elderly = 8-22 mg%
Urea concentration = BUN x 2.14 Urea:
Molecular mass of urea = 60 Adults = 15-38 mg%
Mass of nitrogen = 14(2) = 28 (nitrogen mass in urea) SI Unit = mmol/L
Example: Conversation Factor: mg% mmol/L = 0.167
BUN = 18 mg%
Urea (mg%) = 18 mg% x (60/28) = 38.52 mg% Clinical Significance of Urea Determination
 Azotemia
o Biochemical derangement characterized by an elevated urea in plasma Methods of Creatinine Determination:
o No signs & symptons of disease I. Chemical Methods
Uremia A. Based on the Jaffe's reaction
o There is an elevated level in plasma B. Based on 3,5 dinitrobenzoate reaction
o Manifestation of signs & symptoms of disease II. Enzymatic Methods
o More severe condition A. Creatinine iminohydrolase method
Azotemia Uremia B. Creatinine hydrolase method

Three Types of Azotemia: Chemical Methods

1. Pre-renal Azotemia A. Based on the Jaffe reaction
o Causes are before the kidneys due to:
1. Manual method
High protein intake 2. Automated method - kinetic rate principle
protein catabolism - e.g. muscle wasting Reagents: Alkaline picrate
Protein reabsorption after GIT hemorrhage o Picric acid (yellow)
perfusion of the kidney o NaOH
Mild dehydration Principle Involved:
Acute tubular necrosis o Creatinine + Alkaline picrate Creatinine picrate (orange to red)
2. Renal Azotemia tautomer
o Due to renal impairment (kidney disease) o Absorbance read at 485-520nm (follow Beer-Lambert's Law)
Glomerulonephritis Quasi Creatine Chromogens (QCC)
Pyelonephritis o Interferes with creatine determination
3. Post-renal Azotemia Ascorbic acid
o Associated with abnormalities after the kidneys Proteins
Blockage due to stones Glucose
Acetoacetate - a ketone body
Plasma urea concentration requested together with Plasma creatine Pyruvate
BUN : Creatinine :: 12-20 : 1 Cephalosporins
Guanidine
CREATININE & CREATINE PSP - phenolsulfopthalein
Creatine - derived from 3 amino acids (ARG, GLY, MET) synthesized in the liver, BSP - bromosulfopthalein
kidneys & pancreas then transported to muscle & brain. Non protein substance o Lloyd's reagent/Fuller's earth
of the blood. Aluminasilicate which binds to all QCCs (to be removed) except
Creatinine
1-2 % of creatine in muscle converted to creatinine daily o Gum ghatti
Creatine Creatinne Added to eliminate turbidity formation
B. Based on the 3,5 dinitrobenzoate reaction
Creatinine - anhydride of creatine; end-product of metabolism Creatinine + 3,5 DNB orange-red colored complex
o Creatinine is measured before and after heating
o Cb - Ca = Creatine
Enzymatic Methods
o In the muscle, creatine is phosphorylated

A. Creatinine iminohydrolase (creatininase) method
o Creatine + ATP > Creatine-PO4 + ADP
Creatinine > N-methylhydantoin + NH4+
o Creatine-PO4 Creatine Creatinine anhydride NH4 is measured
 Van Slyke Method is used
B. Creatinine hydrolase method Methodology
. h I. Phosphotungstic Acid Methods (PTA)
Creatinine > Creatine (measured using coupled enzyamtic
1. Folin - uses phosphotungstate
reaction) 2. Brown - uses phosphotungstate

Creatine + ATP > Creatine-PO4 + ADP 3. Newton - uses arsenotungstate

. 4. Archibald (Modification of Kern-Stransky Method)
Phosphoenolpyruvate > Pyruvate
Uses phosphotungstate (Protein precipitant and color
ADP ATP developer)

Pyruvate > lactate Uses 0.5 N NaOH (destroys substances in PTA rxn -- ascorbic

+ acid, glutathione, ergothionine, cysteine and other SH-
NADH + H NAD+
containing compounds
Monitored at 340 nm A concentration of creatinine in sampe
5. Henry method
Same principle as #4 but uses 14% Na2CO3
Normal Values:
6. Caraway - uses phosphotungstate and sodium carbonate
Non-specific methods (using Jaffe reaction)
Females = 0.7-1.3 mg%
Principle Involved:
Males = 0.9-1.8 mg%
Uric acid + Phosphotungstate/Arsenotungstate Tungsten blue (610-715 nm)
Specific methods (using Jaffe's reaction; use of Lloyd's reagent -
Uric acid Allantoin
aluminum sulfate)
Allantoin + PTA/ATA Tungsten blue
Females = 0.12-1.22 mg%
Males = 0.53-1.0 mg%
II. Uricase Method
Specific for uric acid
SI Unit = use conversion factor 88.4 (mol/L)
Reaction Catalyzed:
For Renal impairment
O2 CO2
Process of urine formation: 22
1. Glomerular filtration Urate > Allantoin

2. Tubular reabsorption
3. Tubular secretion Uses the principle of differential spectrophotometry
Urate - with significant A at 282 - 292 nm
Creatinine Clearance Allantoin - has no A
Volume of plasma freed or cleared of creatinine per unit of time Change in A before and after addition of uricase concentration of
Creatinine is completely filtered by the kidneys uric acid in sample
Neither reabsorbed nor secreted
Normal Values:
Way of determining glomerular filtration rate (gold standard)
PTA method
URIC ACID Female = 3.0 - 6.5 mg%
End product of purine catabolism in man Male = 4.5 - 8.2 mg%
For diagnosis of Gout Uricase method - decreased because it does not measure interfering
Adenine, Guanine - Purines substances
Female = 2.6 - 6.0 mg%
Allantoin - end product of purine catabolism in lower animals Male = 3.5 - 7.2 mg%
 SI = mmol/L
Conversion Factor = 0.059 Xanthine
Xanthine oxidase
Clinical Significance Uric acid
Hyperuricemia = increase uric acid blood
Types: E+CI ECI
1. Essential uricemia - due to overproduction and under excretion of uric
acid LIPIDS
0-2 % = kidneys Insoluble in water but soluble in organic solvent (chloroform, benzene, ether,
Small = GIT etc.)
i. GOUT Nonpolar
ii. Chemotherapy for proliferative diseases Primary storage of energy
Leukemia Simple, Complex, precursor/derived
Lymphoma
Polycythemia Lipid Methodology
Multiple myeloma Lipid Profile Determination
Increased nuclear catabolism Total Cholesterol
Iii. Renal disease TG
2. Secondary hyperuricemia Lipoprotein determination (LDL, HDL)
i. Glycogen Storage Disease Requested to determine risk to CVD
And other genetic enzyme deficiency
Triglyceride Specimen: Plasma or Serum (fasting to avoid false elevation of TG and cholesterol)
Lactic Acid General Procedure: Total cholesterol, cholestery esters & free cholesterol

Compete with uric acid 1. SAPONIFICATION
Urinary excretion o Specimen + Alcoholic KOH (Absolute Ethanol & 33% KOH)
ii. Deficiency in HGPRT o Heat and cool afterwards
a. Hypoxanthine-Guanine Phosphoribosyl Transferase o Formation of soap
(HGPRT) 2. CHOLESTEROL EXTRACTION
Enzyme involved in the salvage pathway for o Mixture from #1 + Ethyl Alcohol - Petroleum Ether mixture (Bloor's
purines mixture); 1:3
Lesch-Nihan Syndrome - a nervous disorder o Bloor's mixture - extract cholesterol from saponified sample
characterized by mental retardation and self- o Centrifuge and extract upper petroleum ether layer
mutilation 3. EVAPORATION TO DRYNESS
3. Hypouricemia o Extracted petroleum ether layer evaporated to dryness (60C)
Defective reabsorption of uric acid o Too high temperature will lead to charring
Renal disease -- e.g. Fanconi syndrome o Residue is left behind
Overtreatment with allopurinol 4. COLOR REACTION
4. Gout o Reagent used: Liebermann-Burchard color reagent (color-developing;
Treated by Allopurinol react with cholesterol found In the residue)
Adenine /Guanine a. Acetic acid anhydride - 20 parts
b. Concentrated H2SO4 - 1 part
c. Glacial Ac - 10 parts

 o Color formed is from yellow to green depending on the oxidation of the Triglyceride(Triacyl Glycerol) Determination
color-developing reagent General Procedure:
1. Extraction of TG from serum/plasma
A. Color Reactions for Cholesterol: o Uses
o Done in the dark, if not will lead to false decrease in concentration Isopropanol or chloroform - methanol mixture & an adsorbent -
silicic acid or zeolite (sodium aluminum silicate)
1. Liebermann-Burchard Reaction 2. Conversion of carious forms of TG into one form
Cholesterol Cholesta-hexaene sulfonic acid (green to yellow o Triglycerides
h
> Free glycerol

@ 620 nm)
o Short or medium or long chain TG which is dependent on the length of
SO3- will oxidize phenantrene rings, leaving behind double
fatty acids
bonds
3. Determination of Glycerol using
Will go on until 6 double bonds will be formed (hexaene)
Evelyn & Malloy made a method of total cholesterol determination o Glycerol > Formaldehyde
based on the LB reaction Formaldehyde + Chromotropic acid (color developing reagent) Pink
Gold standard derivative (565 nm)
2. Zak Reaction a. Steps above + NH4 + Acetyl acetone
h
> Lutidine derivative
Cholesterol Tetraene derivative (reddish violet)

Fe+++ using FeCl3 (Yellow at 410 nm)

3 double bonds will be formed through oxidation c. Participation of Glycerol

3. Salkowski Reaction Glycerol + ATP > Glycerol-3-PO4 + ADP

Almost similar to Zak reaction Phosphorylation
Produces three double bonds ADP + Phosphoenol pyruvate

> ATP + Pyruvate

Transfer of Phosphate from Phosphoenol pyruvate to ADP
Disadvantages of Methods using the above Reactions
Pyruvate + NADH+H+ (reduced) > Lactate + NAD+
1. Corrosive nature of the reagents h
2. Difficulty in automation (oxidized)
3. Instability of the final color Measures the decrease in absorbance at 340 nm, which is
4. Problems with standardization proportional to the concentration of the free glycerol in the
sample, which is then proportional to the TG

B. Enzymatic Method for Cholesterol d. Glycerol + ATP > Glycerol-3-PO4 + ADP

h Glycerol-3-PO4 + NAD > Dihydroxyacetone PO4 + NADH
4 h
Cholesteryl ester > Free cholesterol + Fatty acid anion +
+H
h
Cholesterol > 4-Cholesten-3one + H2O2 H+ + NADH + INT
h
> Formazon(red at 500 nm) +

( h)
2 H2O2 + 4-Aminoantipyrine + Phenol > Quinoneimine dye (505 nm) + NAD +

H2O
Normal Values
o Standard conditions: pH 6.7 at 37C 30-160 mg/100 mL (young adults) or 0.34-1.81 mmol/mL up to 210 mg/100 mL
o Available in a kit (CHOD-PAP) (older people)
Conversion factor to SI (mmol/mL) = 0.0113
Normal values:
150 - 250 mg/100 mL or 3.9-6.5 mmol/L; now 100-200 mg/100 mL Clinical Significance
Conversion factor to SI (mmol/L) = 0.026 Hypercholesterolemia - cholesterol level above normal
 Hypertriglyceridemia - TG level above normal Four Groups of Structural Abnormalities (Hemoglobinopathies)
Hypocholesterolemia - levels below normal 1. Amino Acid Substitutions
Hypotriglyceridemia o HbS
Defect in the coding of the -chain in the 6th amino acid GLU into
LDL-Cholesterol VAL
HDL-Cholesterol o HbC
o HbD
General Methods o HbE
1. Ultracentrifugation o HbO
o NaCl specific gravity = 1.062 o HbG
o High speed centrifugation o Most common (several hundreds)
o Chylomicrons - 0.091 2. Amino Acid Deletion
o VLDL - <1.006 o Deletions of three or multiples of three nucleotides in DNA
o LDL - 1.006 - 1.063 o Example Hb Gun Hill
o HDL - 1.063 - 1.21 3. Elongated globin chains resulting from chain termination, frame shift, or other
o Determine cholesterol content of LDL & HDL L-B reaction mutations
2. Electrophoresis o Hb Constant Spring
o VLDL - pre lipoprotein 4. Fused or Hybrid chains resulting from non-homologous crossover
o LDL - lipoprotein o Hemoglobin Lepore - (delta-beta globin chain)
o HDL - lipoprotein o Hemoglobin Kenya - (gamma-beta globin chain)
o Densitometry - quantify
2/3 of hemoglobinopathies have an affected beta chain
HEMOGLOBINOPATHIES & THALASSEMIA May be clinically silent or cause severe damage
Hemoglobin
A quaternary protein found inside the rbc's Thalassemias (Hemoglobin Quantitative Defects)
Metalloprotein - Fe++ or Fe+++ There is a defect in the rate of synthesis of one or more of the Hb chains, but
Chromoprotein the chains are structurally normal
It is made up: Decreased of absent chain synthesis are caused by gene deletions or point
o Non-protein component = HEME mutations
o Protein component = GLOBIN
4 polypeptide chains Two Common Types:
o Each subunit is made up of 141 amino acids 1. Alpha Thalassemia - defective production of alpha chains
o Each subunit is made up of 146 amino acids o Hb Bart - 4
o Hb H - 4
Hemoglobinopathy - a group of diseases resulting from genetic defects in the Hydrops fetalis in babies
STRUCTURE of hemoglobin 2. Beta Thalassemia - failure to produce normal amounts of globin chains
o Thalassemia major/Cooley's Anemia - homozygous
Thalassemias - disorders resulting from defects in RATE & QUANTITY of Globin Deletion, mutation
production o Thalassemia minor - heterozygous
mRNA
Genetic Locus for Globin Chains:
Chromosome #16 - contains the genes Screening Tests for Hemoglobinopathies
Chromosome #11 - contains the non- genes (, , ) 1. Dithionate Solubility Test for HbS
 o When hemoglobin S is in the deoxygenated state it is insoluble, a PBG - Watson-Schwartz & Hoesch Test
precipitate is produced in the test o Uses Erlich's Reagent (Para-aminodimethyl azobenzene)
2. Electrophoresis o Yellow or green color is indicative of PBG
o Cellulose acetate Porphyrins - Red fluorescence when viewed with ultraviolet light
o Agar
3. Chromatography Catabolism of Heme (pictures from Dana)
4. Genetic studies A. Formation of Bilirubin
B. Conjugation of Bilirubin in the Liver
Biosynthesis of Porphyrin Heme (picture from Dana)
h h ELECTROLYTES
Glycine + Succinyl CoA > -amino--ketoadipate > -
4 2 Charged particles or ions capable of conducting electrical current
aminolevulinate (ALA)
h
Two molecules of -aminolevulinate > porphobilinogen (first precursor Cations - positively (+) charged particles
22
pyrrole) Anions - negatively (-) charged particles
Ferrochelatase catalyzes the insertion of Fe++ to protoporphyrin III to produce
heme Major Cations:
1. Sodium (Na+) - major extracellular cation
Clinical Significance: 2. Potassium (K+) - major intracellular cation
Compounds of Heme synthesis that are clinically significant: 3. Calcium (Ca2+)
1. Delta-aminolevulinic Acid 4. Magnesium (Mg2+)
2. Porphobilinogen (PBG)
Porphyrin Compunds: Major Anions:
3. Protoporphyrin (PROTO) 1. Chloride (Cl-) - major extracellular anion
4. Uroporphyrin (URO) 2. Bicarbonate (HCO3-)
5. Coproporphyrin (COPRO) 3. Phosphate (PO42-) - major intracellular anion
Increase levels in biologic fluids indicates abnormality in heme synthesis 4. Sulfate (SO42-)
5. Protein anions (Proteins-)
Porphyrias - inherited or acquired enzyme deficiencies resulting in 6. Organic acids
overproduction (accumulation) of heme precursors in bone marrow
(erythropoietic porphyrias) or the liver (hepatic orphyrias) Major Function: Acid-Base balance

Increase ALA or PBG or both - associated with neuropsychiatric symptoms, Methodology

abdominal pain, vomiting, constipation, tachycardia, HPN, fever, leukocytosis & Specimen: Serum or Plasma
paresthesias Glasswares: Must be acid-washed & rinsed with deionized water

Increase PROTO, URO or COPRO - photosensitivity, blisters, excess facial hair, Sodium (Na+) and Potassium ions (K+)
hyperpigmentation & other cutaneous signs 1. Flame Emission Photometry - reference method (gold standard)
Ex. Porphyria cutaneous (PCT) Na+ = yellow
Hepatoerythropoietic porphyria (HEP) K+ = violet
Congenital erythropoietic porphyria (CEP)
Erythropoietic porphyria (EP) Chloride Ions (Cl-):
1. Specific Tests for Chlooride Ions
Screening Tests: a. Mercuric Nitrate Titration Method (Schales & Schales Method)
 A titrimetic method Potassium (K+) = 3.5-5.0 mEq/L or mmol/L
Principle Involved: Magnesium (Mg2+) = 1.5-2.5 mEq/L or mmol/L
Cl- (specimen) + Hg2+ (Hg(NO3)2 HgCl2 Chloride (Cl-) = 96-106 mEq/L or mmol/L
s-diphenylcarbazone (fresh, colorless) = indicator (faint pink to violet Calcium (Ca2+) = 4.2-5.2 mEq/L or mmol/L
(end-point) Bicarbonate = 24-28 mEq/L or mmol/L
Phosphorus = 1.0-1.5 mEq/L or mmol/L
Volume of Hg(NO3)2 used to reach end-point = amount of Cl- present in
specimen

b. Cotlove Chloridometer
Semi-automated method
Can also be used for CSF chloride
Principle Involved:
Sample + nitric acid-acetic acid solution Titration Vessel Two
Pairs of Electrodes
First pair = contains a Ag wire (generates Ag+ at a constant rate)
Second pair = acts as sensing electrode detecting changes in the
electric-conductivity of the solution

Greater electrical change, greater amount of chloride ions

Calcium & Magnesium

1. Atomic Absorption Spectrophotometry (AAS)
o Reference method for calcium ions
2. Titan Yellow Method
o Colorimetric method for magnesium ions

Bicarbonate Ions (HCO3-):

1. Gasometric Analysis - Van Slyke Method

Anion Gap:
Used in checking the accuracy of electrolyte determination
Cannot diagnose any disease but its purpose is to check the accuracy and
precision of the electrolyte determination
AG = ([Na+] + [K+]) - ([Cl-] + [HCO3-])
Normal value = difference of 12-18 mmol/L

Reporting Values of Electrolyte Determination

SI System:
For univalent ions (Na+, K+, Cl-) = mmoles or milliequivalents/L
/
For divalent ions (Ca2+, Mg2+) = mmol/L or
2 ()
Normal Values:
Sodium (Na+) = 136-145 mEq/L or mmol/L