OUTLINE 
1. Spectrophotometry (p. 1) 
2. Fasting Blood Sugar (p. 2) 
3. Total Blood Cholesterol (p. 5) 
 
SPECTROPHOTOMETRY 
● a method in which it measures of how much a 
chemical substance absorbs or transmits light   
● the intensity of light is measured as a beam of   
light passes through sample solution  COLORIMETER VS. SPECTROPHOTOMETER 
● The resultant color observed is determined by the   
proportion of different wavelengths remaining in    Colorimeter  Spectrophotometer 
the beam emerging from the solution  (Filter 
  Photometer) 
PRINCIPLES OF SPECTROPHOTOMETRY  Light source  LED, Fixed  Lamp (Tungsten, 
● Works similarly with titrimetric, gravimetric, and  wavelength  Xenon, Deuterium), 
other procedures in analytical  wavelength range 
chemistry  Wavelength  Color filter, Fixed  Monochromator, 
o Detects the amount of analyte present in  selector  wavelength  wavelength range 
a given sample  Portability  Stationary parts,  Moving parts, 
● Used to measure how much a substance absorbs  light weight, good  heavier, good for 
light by measuring the  for field use  bench use. 
intensity of light as a beam of light passes   
through a sample solution  The DR 1900 can 
o Measurement is used to identify the  be used in the 
amount of a known substance by  field, it is lighter 
determining  and battery 
its concentration  operated but still 
  has some moving 
ESSENTIAL PARTS OF A SPECTROPHOTOMETER  parts. 
1. Light source  Parameters  Single or limited  Numerous 
2. Wavelength selector (monochromator)  number of  parameters 
3. Sample compartment  parameters  determined by the 
4. Detector   determined by  wavelength range.  
fixed 
wavelengths 
 
IMPORTANCE 
● This measurement can be used to measure the 
amount of a known chemical substance 
● The range of the wavelength(or frequency) within 
which absorption occurs depends upon the nature 
of absorbing material 
 
  Light 
● The type of light source will depend on the 
desired wavelength.   
● Light in the visible range (350 nm - 800 nm) is 
best supplied by a tungsten filament lamp.   

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BIOCHEMISTRY: Laboratory Discussion
 ● The wavelength selector splits the light spectrum  an increase in the length of the solution 
into its component colors and selects a narrow  traversed. 
band of this spectrum.     
● Either a prism or a grating may be used as a  Beer-Lambert’s Law 
dispersing element to split the light.    ● “The light is absorbed in direct proportion to the 
● The selected light passes into the sample  concentration of the absorbing species in solution 
compartment, through the sample (and  and the length of the path traversed by the light.” 
cell/cuvette) and to the detector   
(photomultiplier).  By measuring transmittance 
with (I) and without (I​o​) the sample present, 
absorbance can be determined.  This is how a 
single beam spectrophotometer operates. 
 
Factors Affecting Absorption:  

 
 
Sample Problem: 

 
 
Optical Density 
 
 
LAWS OF ABSORPTION OF LIGHT 

 
● The main advantage of using Optical Density is 
that OD is directly proportional to concentration 
when the length of the light patch remains 
constant. Thus, when OD is plotted against c, a 
straight line is obtained. 
● To convert a value from percent transmittance 
(%Ts) to absorbance (A): 
 
Absorbance (A) = 2 - log(%Ts) 
 
  ● To convert a value from absorbance to percent 
Beer’s Law  transmittance: 
● The intensity of a monochromatic light decreases   
exponentially as the concentration of the  %T = antilog(2 - absorbance) 
absorbing material increases.   
   
Lambert’s Law   
● States that the intensity of a ray of   
monochromatic light decreases exponentially with 

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BIOCHEMISTRY: Laboratory Discussion
  
Glucose 
 
Oxidase 
 
ENZYMATIC DETERMINATION OF FASTING BLOOD SUGAR  ● Enzyme should be added last. At the same time, 
● The  most  important  hexoses  are  D-glucose,  mix each tube gently by inverting the test tube 
D-galactose,  and  D-fructose,  but  the  principal  several times. 
sugar circulating in the bloodstream is glucose.   ● Incubate all tubes in a water bath at 37°C for 45 
● Lactose  (glucose  and  galactose)  and  sucrose  minutes or at room temperature for one hour. 
(glucose  and  fructose)  are  important  (​Note​: Avoid exposure to direct sunlight or bright 
disaccharides.  light) 
● Carbohydrates  are  measures  in  whole  blood,  ○ It is necessary to avoid exposure to light 
serum or plasma.   and prevent excessive shaking to prevent 
● The  concentration  of  glucose  in  blood  is  normally  the autoxidation (or photoxidation) of the 
controlled  within narrow limits by many hormones,  enzyme. (Color of the mixture: peach) 
the  most  significant  of which, insulin, is produced  ● Read at 460 nm using the reagent blank to set 
by the endocrine pancreas.  the spectrophotometer to zero. 
  ● Test samples must be read within 30 minutes 
REAGENTS  after incubation. 
1. Plasma Glucose Oxidase​​ (PGO)​​ Enzyme   
(pre-weighed capsule): the enzyme solution is  Calculation: 
prepared as follows: 
○ add the contents of one capsule to 100 
ml distilled water in an amber colored 
bottle and invert the bottle several times 
with gentle shaking to dissolve contents. 
2. O-Dianisidine Dihydrochloride Color Reagent​​: the 
color reagent solution is prepared as follows:   
○ dissolve 50 mg of O-dianisidine   
dihydrochloride in 20 ml distilled water.  RESULTS 
3. Combined Enzyme-Color Reagent Solution​​:  From Group 18A’s data: 
○ To 100 ml of enzyme solution, add 1.6 ml 
of color reagent solution. Mix by inverting 
several times with mild shaking. Store in 
a brown bottle. 
4. Standard Glucose solution​​ = 200 mg/dl    
Note: Molecular weight of Glucose is 180.1446 g/mol 
 
Interpretation: 
- The subject’s blood glucose level falls within the 
normal range in this case. 
 
PROCEDURE 
 
Content  Tube 1  Tube 2  Tube 3   
 
Blank  Standard  Sample  CHEMICAL REACTIONS INVOLVED 

Water  0.02 ml     

Glucose    0.02 ml   

standard 
 
Serum      0.02 ml  ● Gluconolactone is simply an intermediate 
● Hydrogen peroxide has a direct linear relation with 
Combined  3.0 ml  3.0 ml  3.0 ml  the amount of glucose oxidized 
Enzyme  ● Colorless O-Dianisidine is in its reduced form 

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BIOCHEMISTRY: Laboratory Discussion  
 ● Hydrogen peroxide oxidizes the O-Dianisidine with   
peroxidase, which catalyzes the cleavage of   
peroxide and the oxygen released is received by  STUDY QUESTIONS 
O-Dianisidine, thus becoming oxidized.  1. What are the recent criteria in diagnosing 
● The intensity of the color is proportional to the  diabetes mellitus according to American Diabetes 
amount of glucose present.  Association (ADA)?  
  - The ​A1C test​​ measures your average blood 
  glucose for the past 2 to 3 months. The 
KEY POINTS IN FBS DETERMINATION  advantages of being diagnosed this way are that 
● The diagnosis of diabetes requires a fasting  you don’t have to fat or drink anything.  
plasma glucose of ≥126 mg/dl (7.0 mmol/L) on  - Diabetes is diagnosed at an A1C 
at least two occasions or a casual or random  of greater than or equal to 6.5%.  
plasma glucose level (or 2-hr post-glucose load 
level) of ≥200 mg/dl (11.1 mmol/L). 
● Oral Glucose Tolerance Tests​​ (OGTT) should be 
performed to diagnose gestational diabetes. 
(between 24th to 28th week of pregnancy) 
○ A certain level of insulin resistance is 
normal during pregnancy. The placenta   
produces insulin-blocking hormones that  - Fasting Plasma Glucose (FPG)​​ - this test checks 
makes sugar stay in blood after a meal.   your fasting blood glucose levels. Fasting means 
● Whole blood capillary glucose values obtained  that after not having anything to eat or drink 
with point of care devices are useful for the  (except water) for at least 8 hours before the test. 
detection of hyperglycemia and hypoglycemia in  This test is usually done first thing in the morning, 
individuals with diabetes, and help monitor and  before breakfast. 
direct therapy. They SHOULD NOT be used to  - Diabetes is diagnosed at fasting blood 
diagnose diabetes or hypoglycemic disorders. To  glucose of greater than or equal to 126 
establish these diagnoses, confirmation with  mg/dl 
laboratory measures of plasma glucose are 
essential because of their greater accuracy.  

 
- Oral Glucose Tolerance Test (OGTT)​​ - the OGTT is a 
  two-hour test that checks your blood glucose 
● HbA1C levels should be performed every 3-6  levels before and 2 hours after you drink a special 
months in individuals with diabetes to monitor  sweet drink. It tells the doctor how your body 
glycemic control using a certified method.  processes glucose. 
Reliability and accuracy are diminished in the  - Diabetes is diagnosed at 2 hour blood 
presence of shortened red cell survival, lower  glucose of greater than or equal to 200 
mean blood cell age or need for transfusions as  mg/dl. 
seen with certain hemoglobinopathies and 
hemolytic conditions, as well as with uremia.  
● Commonly  used  strips  and  tablets  for  ketone 
testing  use  sodium  nitroprusside,  which  does not 
detect  beta-hydroxybutyrate.  Since 
beta-hydroxybutyrate  levels  are  high  in  diabetic 
ketoacidosis  (DKA)  and  fall  with  treatment,   
whereas,  acetoacetic acid and acetone levels rise  - Random Plasma Glucose Test 
with  treatment,  these  strips  are  not  useful  to  - This test is a blood check at any time of 
monitor therapy.   the day when you have severe diabetes 
  symptoms. 
  - Diabetes is diagnosed at blood 
  glucose of greater than or equal 
  to 200 mg/dl.  

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BIOCHEMISTRY: Laboratory Discussion 
 
2. What is the role of oral glucose tolerance tests (OGTT) 
in diagnosing diabetes mellitus? 
- Oral Glucose Tolerance Test 
- Measures the ability of the body’s cell to 
absorb glucose after consuming on 
specific amount of sugar. 
- Use fasting blood sugar levels and 
hemoglobin A1C.  
- Type 1 DM - after the intake it quickly 
diagnose type 1 because of high blood 
sugar levels and symptoms. 
- Gestational Diabetes​ - females who don’t   
have diabetes have a possibility to high   
blood sugar because of pregnancy.   5. What is the role of glycosylated hemoglobin in the 
  management of patients with diabetes mellitus? 
3. How is an OGTT carried out in:  Glycosylated hemoglobin (HbA1c) 
a. Adult individuals:  - A level on which reflects the patient’s average 
Steps:  blood sugar for 2 months. 
- Fasting blood sugar level test  - By monitoring the level of HbA1c, practitioner can 
- Drink 8 ounces of a syrup glucose  easily manage the diabetes mellitus and to 
solution (75 grams of sugar)  prevent its complications. 
- Wait for 2 hours (1 and 2-hour mark)   
b. Pregnant high-risk females:  SERUM CHOLESTEROL DETERMINATION 
Steps:  ● The blood is the body’s main means of 
Part 1  transporting nutrients 
- Fasting blood sugar level test  ● Understanding the normal or physiologic blood 
- Drink a solution with 50 grams of sugar  composition allows scientists and healthcare 
- Wait for 1 hour  professionals to determine any types of 
Part 2 (if necessary)   pathologies in the patient. 
- Consume syrup glucose solution (100  ● Cholesterol, despite being extremely hydrophobic, 
grams of sugar)  can be transported through the blood through 
- Draw of blood after 1, 2, 3-hour marks  lipoproteins 
after intake.  ○ Cholesterols are esterified to allow better 
- Determine blood sugar level samples  packaging inside the lipoproteins, 
  enhancing the transport mechanism 
4. What is glycosylated hemoglobin (HbA1C)? How is it  efficiency 
formed? Can you show the chemical reaction of its  ● Serum cholesterol level is a measurement of 
formation?  certain elements in the blood which includes the 
- Glycosylated hemoglobin (HbA1c) is the most  HDL, LDL, and Triglycerides. 
abundant minor hemoglobin component in human  ● LDL – “bad cholesterol” 
erythrocytes.  ● HDL – “good cholesterol” 
- It is the primary protein used for glycemic control,   
involving a non-reversible process of attaching  CLINICAL APPLICATION OF SERUM TOTAL CHOLESTEROL 
D-glucose to a specific amino-terminal site of the  ● Increased cholesterol 
β-chain of hemoglobin.  ○ Diabetes mellitus, nephritis, and 
- This process comprises three stages of  nephrotic syndrome, early liver cell 
non-enzymatic reactions, known as the ​Maillard  disease, and hypothyroidism 
reactions​. The first stage of the reaction is the  ● Decreased cholesterol 
fast, but reversible, formation of an aldimine  ○ Hyperthyroidism, ACTH, and cortisone 
base, and the second stage is the considerably  therapy 
slower and non-reversible formation of HbA1c.  - Serum total cholesterol levels correlate positively 
The final stage of the reaction is the formation of  with the risk of a person getting coronary heart 
hemoglobin advanced glycation end product  disease.  
(Hb-AGE).   
 
 

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BIOCHEMISTRY: Laboratory Discussion
 PRINCIPLE (ENZYMATIC DETERMINATION)  ● Mix  the contents gently by inverting the test tubes 
● The cholesterol assays involves sequential  several times. 
enzymatic reactions. Cholesterol esterase  ● Incubate  all  tubes  in  a  water  bath  at  37°C  for  5 
hydrolyzes cholesteryl esters to cholesterol and  minutes. 
free fatty acids.  ● Read  at  500  nm  using  reagent  blank  to  zero  the 
● Cholesterol oxidase then oxidizes cholesterol to  instrument. 
form cholest-4-en-3one and hydrogen peroxide.   ● Test  samples  must  be  read  within  30  minutes 
● Peroxidase catalyzes the hydrogen peroxide  after color development.  
oxidation of 4-aminoantipyrine with subsequent   
coupling to phenol.  PRINCIPLES BEHIND THE METHODOLOGY 
● The end product is a quinoneimine dye that   
absorbs at 500 nm wavelength. The color 
determined at 500 nm is directly proportional to 
serum cholesterol determined.  
   
METHODOLOGY  ● Enzyme used can also be alternatively referred to 
as cholesterol ester hydrolase 
● Separate the cholesterol ester to its constituent 
cholesterol and fatty acid 
 

 
REAGENTS   
a) Cholesterol reagent​​: This contains the following  ● A radical in the form of hydrogen peroxide is 
mixtures:  formed in this reaction 
i) Cholesterol esterase ≥ 125 U/L  ● The peroxide can be used to indirectly determine 
ii) Cholesterol oxidase ≥ 200 U/L  the amount of cholesterol 
iii) Peroxidase ≥ 1000 U/L 
iv) 4 aminophenazone: 0.5 mM 
v) Phenol: 15 mM 
vi) Phosphate buffer pH = 0.1 M 
vii) Sodium cholate = 3.74 mM 
 
b) ​Cholesterol standard​​ = 200 mg/dl in alcohol or 5.17   
mmol/L in alcohol (2 mg/ml)   ● The product quinoneimine dye is a good reagent 
  used in spectrophotometry capable of absorbing 
Molecular weight of cholesterol = 386.7 g/mol  light depending on its intensity/concentration 
  ● Its intensity is correlated with the amount of 
PROCEDURE  cholesterol in the sample 
   
SUMMARY OF ENZYMATIC REACTIONS 
  Tube 1  Tube 2  Tube 3 
● 1  mole  of  cholesterol  ester  →  1  moles  of 
cholesterol and 1 mole of fatty acid 
Blank  Standard  Sample 
● 1  mole  of  cholesterol  →  1  mole  of 
Water  0.02 ml      cholest-4-en-3one  and  1  mole  of  hydrogen 
peroxide 
Cholesterol    0.02 ml    ● 2  moles  of  hydrogen  peroxide  (reacted  with 
standard  4-aminoantipyrine  and  phenol)  →  1  mole 
quinoneimine dye and 4 moles of water 
Serum      0.02 ml   
2 moles of cholesterol ester: 1 mole of quinoneimine dye 
Cholesterol  2.0 ml  2.0 ml  2.0 ml  ● Darker tint/ coloration = more quinoneimine = 
reagent   more cholesterol esters in sample 
● Darker tint = higher attenuation = higher 
● Add the cholesterol reagent last  absorbance 

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BIOCHEMISTRY: Laboratory Discussion 
 RESULTS AND DISCUSSIONS  ● Apolipoproteins​​ – are the protein moieties of a 
lipoprotein; they can function as enzyme cofactors 
and can act as ligands for interaction with 
lipoprotein receptors. 
 

 
From Group 19A’s data: 
   
 
2. How is the Friedewald formula calculated? 
 
● The Friedewald formula is currently the main 
method for evaluating low-density lipoprotein 
cholesterol (LDL-c). Recently, many limitations 
have emerged regarding its use, including 
patients with triglyceride levels ≥400  mg/dL, 
diabetes mellitus, and kidney or hepatic chronic 
diseases. 
 
 

 
 
3. What are the target cholesterol values in a 
  complete lipid profile? 
 
INTERPRETATION 
Normal Laboratory Value​: Serum Cholesterol = 150-220 
mg/dL (3.9 - 5.72 mmol/L) 
 
● Obtained value for serum total cholesterol in this 
problem is 137.7 mg/dL or 3.5609 mmol/L 
● The value obtained is slightly below the normal 
range 
● Low total cholesterol levels are generally 
preferred, but extremely low levels (Total   
cholesterol less than 120 mg/dL, LDL less than   
50 mg/dL) are associated and/or increase the  REFERENCES 
risk of developing health problems  1. Group Reports (18A, 19A, 20A) 
Possible Causes:  2. Laboratory Manual 
● Family history of the condition 
TRANSCRIBERS 
● Poor/Unhealthy diet 
1. TRANSCRIBER: “tagabuhat ng grupo” 
● Sedentary lifestyle 
2. SUBTRANSHEAD: AT 
● Handling errors 
 
STUDY QUESTIONS 
De La Salle – Health Science Institute College of Medicine 
1. What are apolipoproteins and what is their role in 
Batch Twenty Twenty-Two 
lipid metabolism? Can you enumerate the four 
 
major classes of apolipoproteins and differentiate 
“non sibi sed omnibus” 
them? 
   
 

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BIOCHEMISTRY: Laboratory Discussion