5.

02 Dietary Variation Plenary

BCH
LE 5
Aileen Carlos, MD | February 26, 2019 TRANS 2

potatoes, fruit & emphasis on

OUTLINE butter purine-rich foods
I. Overview on Blood and Urine Parameter Omit dish and meat Avoid fruits &
II. Protocol starchy foods
a. Preparation Butter, margarine, Brain, Fish, Beef, Anchovies
b. Experiment proper breads, cereals, Pork, Poultry, Kidney
III. Blood Parameters cheese, eggs. Shellfish, liver
IV. Urinary Parameters All kind of fats vegetables : Sardine
fruits, milk, sugar & Asparagus, Dry Sweetbread
sweets vegetables bean,s lentils such Meat extracts
OBJECTIVES
except lentils, as mushroom, Gravy
1. To see the effects of certain diets on blood and urine
asparagus & bean spinach Oatmeal
constituents.
2. To study the effects of specific diets based on the
analysis of biochemical parameters shown below. A. PREPARATION FOR THE EXPERIMENT
● Two days before the experiment
→ Start of biochem diet
I. OVERVIEW ON BLOOD AND URINE PARAMETER
● Student volunteers will keep a careful record of food eaten at
each meal
BLOOD URINE
● One day before the experiment
Urea Nitrogen Volume
→ During this period urine will be collected over a 24-hour
Uric Acid pH
period starting on the following morning on the day of the
Total Protein Specific Gravity
experiment
Creatinine Titratable Acidity
→ Toluene was used to preserve the urine specimen
NH3
● On the day of the experiment:
Creatinine Content
→ Volunteers should minimize intake of fatty food during
breakfast (to prevent cloudiness of the serum due to
increased lipid content)
→ Have blood extracted at Biochem Laboratory

B. EXPERIMENT PROPER (WORKFLOW)

Measurement of Titratable Acidity

● PROCEDURE:
TITRATABLE ACIDITY

place 25 mL urine in 250 mL Erlenmeyer flask

add 5 grams Potassioum Oxalate

add 1-2 drops phenolphthalein solution

Figure 1. Blood Parameters. (Anderson & Cockayne, 2007; p.374) Shake the mixture vigorously for 1-2 minutes

II. PROTOCOL
● Special diets were assigned to 1 volunteer Titrate with 0.1 NaOH until a faint pink color remains
→ Low Protein Diet permanent on forther shaking
→ High Protein Diet ● NOTE : Calculation:
→ High Purine Diet → Y represent the volume of 0.1N NaOH used
→ Y’ represents the volume of urine excreted in 24 hours,
→ The total acidity of the urine excreted in 24 hours or the
Titratable Acidity of the 24 hours urine specimen (x) may
be calculated by means of the following proportion
Low Protein High Protein High Purine
Eat freely of Eat a diet which is Eat a high-protein
vegetables, rice, high in meat diet but with

BCH 1 / 6
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 25 𝑌′
= ● The Stability of Endpoint Reaction: The final color developed in
𝑌 𝑋 the reaction is stable for at least 30 mins. At room temperature;
however direct sunlight should be avoided.
Determination of Ammonia ● CALCULATION of results :
● The neutralized urine in the urinary Titratable Acidity Concentration of STD = 10 mg/dL
determination may be used, and just do the last 2 steps.
● COMPUTATION : 𝐴 (𝑈𝑛𝑘𝑛𝑜𝑤𝑛) 𝑚𝑔
1 mole of 1.1N NaOH is equivalent to 1.7 mg of ammonia. 𝑥 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑆𝑇𝐷 ( )
𝐴 (𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑) 𝑑𝐿
Multiply the number of volume (mL) of 0.1N NaOH. After the 𝑚𝑔
= 𝑈𝑟𝑖𝑐 𝐴𝑐𝑖𝑑 ( )
addition of the formalin by 1.7 and by 4 to get the number of 𝑑𝐿
mgs. + amino acid nitrogen (expressed as ammonia) in 100 mL
of urine examines. Measurement of Serum and Urine Creatinine
Mg of NH3/100 mL urine = mL of 0.1N NaOH (after addition ● Involves the Jaffe Reaction which is based on the reaction of
of formalin) x 1.7 x 4 creatinine with picric acid under alkaline conditions (low pH)
● The reaction produces red colored product ⇀ can be measured
● PROCEDURE by spectrophotometer
DETERMINATION OF AMMONIA ● in this experiment : Heinegard and Tidestorm modification of
Jaffe reaction
● Surfactant and borate are used to reduce interference from
protein and glucose
place 25 mL urine in 250 mL Erlenmeyer flask ● PROCEDURE :
→ Unhemolyzed serum should be used
→ The 24 hour urine should be preserved with toluene. It
add 5 grams Potassioum Oxalate should be diluted X10 with deionized water prior to analysis
add 1-2 drops phenolphthalein solution → Avoid microbial contamination

Serum Creatinine
Shake the mixture vigorously for 1-2 minutes Urine Creatinine

Titrate with 0.1 NaOH until a faint pink color remains permanent Label Test tube unknown/sample
on further shaking

1.5 mL picric acid

Add 10 mL of neutral formalin solution 1.5 mL base reagent
0.1 mL deionized H2O
0.1 mL serum/urine sample

Mix well and titrate with 0.1 NaOH to a permanent pink color
Mix and Incubate 37C x 15 minutes
Spectro 510 nm
Determination of Serum Uric Acid
● PROCEDURE ● CALCULATION (Serum):
SERUM URIC ACID
𝐴 (𝑈𝑛𝑘𝑛𝑜𝑤𝑛) 𝑚𝑔
𝑥 𝐶𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑆𝑇𝐷 ( )
𝐴 (𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑) 𝑑𝐿
2.5 mL URI-ZYME buffer 𝑚𝑔
= 𝑆𝑒𝑟𝑢𝑚 𝐶𝑟𝑒𝑎 ( )
𝑑𝐿
Unknown Test Tube: 0.05 mL serum sample ● CALCULATION (Urine)
𝐴 (𝑈𝑛𝑘𝑛𝑜𝑤𝑛) 𝑚𝑔
𝑥 𝐶𝑜𝑛𝑐 𝑜𝑓 𝑆𝑇𝐷 ( )
Add 0.10 mL of URI-ZYME COLOR REAGENT 𝐴 (𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑) 𝑑𝐿
𝑚𝑔
𝑥 1.67 𝑥 0.01 𝑥 10 𝑥 24 ℎ𝑟 𝑢𝑟𝑖𝑛𝑒 𝑣𝑜𝑙 (𝐿) = 𝑈𝑟𝑖𝑛𝑒𝐶𝑟𝑒𝑎 ( )
𝑑𝐿
Mix gently and allow tos tand at room temperature (20-25C) for
10 minutes NOTE : The factor 1.67 corrects for the calibrator reactivity difference
in an aqueous medium (urine) versus a protein medium (serum). The
factor 0.01 converts mg/dL while the factor 10 adjusts for urine dilution
prior to essay.
Read the absorbance at 520±5 nm wavelength. Use BLANK to
calibrate the spectrophotometer to 0 absorbance
Determination of Creatinine Clearance

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TRANS Maynes, Meriam, Millan, Misagal CORE Mateo, Marasigan, Ng, Ocenar, Ordoñez HEAD Natural
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 𝑚𝑔 𝑚𝐿 A. Urea Nitrogen
𝑈𝑟𝑖𝑛𝑒 𝐶𝑟𝑒𝑎 ( ) 𝑥 𝑈𝑟𝑖𝑛𝑒 𝑉𝑜𝑙 ( )
𝑑𝐿 𝑚𝑖𝑛 ● Urea - major end product of protein nitrogen metabolism; non-
𝑚𝑔
𝑆𝑒𝑟𝑢𝑚 𝐶𝑟𝑒𝑎 ( ) toxic
𝑑𝐿
𝑚𝐿 ● Synthesized in liver from ammonia which is produced by amino
= 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑖𝑛𝑒 𝐶𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 ( ) acid deamination
𝑚𝑖𝑛 ● Passively reabsorbed throughout the nephrons
● Not secreted in healthy individuals; secreted in patients with
Determination of Serum BUN severe renal disease
● Based on hydrolysis of urea in the presence of urease yielding ● 90% eliminated through kidneys
carbon dioxide and ammonium ions
● Ammonium ions + hypochloric acid + nitroferricyanide ⇀ ● 10% eliminated through GIT and skin (e.g., sweat)
chloramines ● Used with plasma creatinine ratio in the UN/creatinine ratio
● Chloramines + salicylate ⇀ quinone chloramines which then ● Experiment is based on the hydrolysis of urea in the presence
reacts with salicylic ion at high pH ⇀ indophenol chromophores of urease yielding CO2 and NH4+ ions.
● Measured photometrically at 630 nm ± 10 nm o (NH2)2CO + H2O → CO2 + 2NH3
● PROCEDURE ● Urine samples: toluene was added which prevents bacterial
growth, conversion of urea to NH3 and preserves urine sample
● Blood samples: Anticoagulants inhibit urease to prevent
SERUM BUN degradation of urea
● Factors affecting plasma urea concentration:
a. Level of protein catabolism – increased tissue breakdown
(e.g. starvation, latter stages of DM) → increased urea
0.5 mL Urea N Color Reagent b. Diet
o High purine/protein diet → increased urea
o Low protein → decreased urea
Unknown Test Tube: 0.01 mL of sample c. Functional status of the liver – impaired liver function
→ ↓ urea synthesis
d. Functional status of the kidney – impaired kidney function
add 0.5 mL of UREA-ZYME REAGENT → ↓ excretion → ↓urea.

Clinical Significance
∙ Azotemia ↔ Uremia
Mix Gently swirling and incubate at 37 C for 5 minutes
(Alternative : 10 minutes at room temperature 22-26C) o Prerenal
o Renal
o Postrenal
Add 2.0 mL of UREA N BASE REAGENT

MIX and Incubate at 37C for 5 minutes (Alternative 10 mins at

room temperature, 22-26C)

Set Wavelength of specthrophotometer 630 nm±10nm

Read and record absorbance

● CALCULATION of results :
𝐴 (𝑈𝑛𝑘𝑛𝑜𝑤𝑛) 𝑚𝑔 𝑚𝑔
𝑥 𝐶𝑜𝑛𝑠𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑆𝑇𝐷 ( ) = 𝑈𝑅𝐸𝐴 ( )
𝐴 (𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑) 𝑑𝐿 𝑑𝐿
Figure 2. Human Urate Homeostasis
2020C trans:
III. BLOOD PARAMETERS 1. Body proteins and dietary proteins through digestion and
absorption, produces amino acids used in the synthesis of
Nonprotein nitrogen compounds pyrimidines. Catabolism of pyrimidines produces ammonia
1. Urea (45%) (NH3).
2. Amino acids (20%) 2. Certain amino acids like glycine, aspartate and glutamate are
3. Uric acid (20%) precursors of purines. In the liver, purines are converted into
4. Creatinine (5%) uric acid (represents degradation product of purines).
5. Creatine (1-2%)
3. In the degradation of amino acids by the process of oxidative
6. Ammonia (0.2%): most toxic
deamination, an amino group is removed and an alpha keto

BCH 3 / 6
TRANS Maynes, Meriam, Millan, Misagal CORE Mateo, Marasigan, Ng, Ocenar, Ordoñez HEAD Natural
Musni
 acid is released. The amino group is then brought to the liver,
where it is converted into urea in the presence of AA Causes of Hypouricemia
aspartate. Urea is eventually excreted in the urine. (major 1. Decreased purine synthesis
end product of CHON nitrogen metabolism) a. Severe liver disease
4. The amino acids can also be converted to creatine and be b. Drugs that inhibit de novo purine synthesis e.g. 6-
converted to creatinine. mercaptopurine, azathioprine
5. Formation and excretion of urea: • Amino acids are 2. Enhanced uric acid elimination
catabolized within cells via oxidative deamination • NH3 is a. Drugs (e.g, allopurinol)
brought to the liver by the blood to be converted into urea via b. Defective renal tubular absorption (Wilson’s disease,
the action of several enzymes + aspartate • Urea is brought Fanconi syndrome, chronic lead or mercury exposure
to the kidneys via the blood and excreted through urine
Gout
● Hyperuricemia + deposition of monosodium urate crystals
B. Uric Acid in joints and tissues
● Final end product of purine nucleoside catabolism, specifically
● Body fluids are supersaturated with urates, crystals precipitate
adenosine & guanosine
as aggregates or tophi in & around joint capsules, tendons,
● Conversion of purines to uric acid occurs primarily in liver then
subcutaneous tissues, cartilage, bone, and in the kidney
to bloodstream
● Many patients with hyperuricemia never develop gout
● Uric acid present in tissues & plasma as urate ions (97%
● Uric acid stones or renal calculi can develop in individuals with
associated with sodium as monosodium urate); pH > 5.57
or without hyperuricemia
● Plasma urates are completely filtered by glomeruli with
● Uric acid crystals can precipitate when urine specimen cools to
subsequent proximal tubular reabsorption & distal tubular
room temperature or is refrigerated (non-significant in most
secretion
routine urinalysis)
● Net result: excretion of 6 – 12% of filtered urates (88 - 94% is
reabsorbed)
● 75% of uric acid eventually excreted in urine C. Total Protein
● 25% secreted into GIT, where it is degraded by bacterial ● N = 5.8 – 8.0 g/dL
enzymes & excreted in feces ● Major plasma fractions
● Based on oxidation of uric acid in the presence of uricase → Albumin
releasing allantoin & hydrogen peroxide. → Globulin
● Hydrogen peroxide reacts with a polyhalogenated benzoic acid ● Clinical Significance
(PHBA) & 4-aminoantipyrine (4-AAP) → Detection of Hypoproteinemia
→ Detection of Hyperproteinemia
● Effect of Diet
→ High purine/protein diet → ↑ total proteins
→ Low protein diet → ↓ total proteins
●
D. Creatine and Creatinine
Table 2. Differences and Similarities
Causes of Hyperuricemia (Increases risk for gout)
Creatine Creatinine
Synthesized in the liver and Waste product of creatine
● Altered Uric Acid Production
pancreas from 3 amino acids and phosphocreatine
1. Increased synthesis of purine precursors
(Arg, Gly, Met) (spontaneous, non-enzymatic)
2. Accumulation of purine precursors due to specific enzyme
pathway deficiencies or defects (Ex. Lesch- Nyhan Amount formed daily is
Acted upon by creatine kinase
Syndrome) equivalent to lean muscle
to form phosphocreatine
3. Increased turnover of nucleic acids mass
− Myeloproliferative syndromes Not reabsorbed anywhere in
− Chemotherapy for leukemias and lymphomas the nephron
Reabsorbed by PCT
Small amount secreted by
● Insufficient Renal Excretion tubules in urine formation
1. Primary Renal disorders Significant role as an index in
Not an indicator of renal
− Acute renal failure renal function, particularly for
dysfunction
− Chronic renal failure monitoring GFR
2. Alteration in renal handling Major source of phosphoryl
− Drug therapy (e.g., salicylates, diuretics) groups for ATP regeneration
− Organic acidemia (ketoacidosis, lactic acidosis) ↑ in skeletal muscle necrosis
− Glycogen storage diseases (e.g., G-6-PD def.) or muscle atrophy
− Toxins (lead, alcohol) Indirectly assessed using
− Toxemia of pregnancy plasma creatine kinase
Similarities
● Transient Hyperuricemia Readily pass glomerular filtration barrier
1. Dehydration – stimulates renal reabsorption of uric acid Essentially cleared from plasma
and ↓ excretion (urine pH < 5.57 → ultrafiltrate is
supersaturated → uric acid crystals precipitate → stones)
2. Ingestion of large quantities of food rich in nucleic acids
Creatinine
(i.e., liver, sweetbreads, anchovies, kidneys and sardines) ● Jaffe Reaction (1886)

BCH 4 / 6
TRANS Maynes, Meriam, Millan, Misagal CORE Mateo, Marasigan, Ng, Ocenar, Ordoñez HEAD Natural
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 → Based on the reaction of creatinine with picric acid under → Ammonia is used to synthesize glutamine, glutamate, and
alkaline conditions that forms a red product measurable at carbamyl phosphate
510nm 1. From carbamyl phosphate, pyrimidines for nucleic acids
● Ideal for monitoring GFR: and urea are synthesized
→ Plasma concentration is maintained at a constant rate → Valuable in evaluating liver function because hepatocytes
→ Completely cleared from plasma at glomeruli are the only cells that contain arginase, an enzyme required
→ Not reabsorbed by tubules for the conversion of ammonia into urea
1. All creatinine cleared is eliminated from the urine ● Clinical significance:
● Usually not affected by non-renal factors (ex. degree of → Hyperammonemia caused by:
hydration, level of protein catabolism) 1. Severe liver disease or liver failure
● Plasma creatinine levels alone are NOT sensitive 2. Inborn metabolic disorders of the urea cycle Hepatic
→ 50-60% of renal function may be lost before plasma encephalopathy
creatinine levels reflect this loss
● Creatinine production is directly related to lean muscle mass
→ Reference intervals vary with gender and age F. Solutes in Urine
● Transient increase in creatinine values can be observed 1. Urea
following severe exercise or high protein ingestion 2. Chloride
3. Sodium
4. Potassium
5. Ammonium
6. Inorganic phosphate and sulfate
7. Creatinine
8. Uric acid

IV. URINARY PARAMETERS

A. VOLUME

Normal Volume = 0.5 – 2L/day

Factors Affecting Urine Volume

● Fluid intake
● Presence of kidney disease
● Drugs (ex. diuretics)
● Weather/temperature (hot vs. cold)
● Emotional stress
→ ↑release of catecholamines (epinephrine and
norepinephrine) -> ↑BP and ↑CO -> ↑renal blood flow ->
↑GFR -> ↑urine volume
Figure 3. Creatinine Synthesis ● Diet
→ High purine/protein: ↑urine volume due to osmotic effect of
urea
E. Ammonia (NH3)
→ Low protein: ↓urine volume
● In the kidneys:
→ Ammonia is generated through the deamination of
glutamine and glutamate B. pH
1. Hydrolysis of glutamine and by trans-deamination as well
as oxidative deamination Normal pH = 5.5 – 8.0 (Ave: 6)
2. Uses Formol Titration Method:
− 4NH4 + 6HCHO -> 4H+ + N4(CH3)5 + 6H2O Factors Affecting pH
− Based on the reaction that takes place when formalin is ● Time of voiding
added to a solution containing ammonium salts → After meals: Alkaline urine (↑pH) due to “alkaline tide” or
o An acid reaction is produced, which is then titrated bicarbonate residues
with standard NaOH using phenolphthalein as an → Long standing or 24-hour urine: Alkaline urine (↑pH) due to
indicator bacterial decomposition of urea to NH3
→ Excreted as the ammonium ion − Hence the ammonia-like odor of urine through bacterial
→ Daily excretion of ammonia and phosphate depends on action
hydrogen excretion in the urine ● Acid-Base Balance
→ NOT for study of renal function → Metabolic acidosis – acidic urine
● In the blood: → Metabolic alkalosis – alkaline urine
→ Majority of ammonia in blood is derived from the GIT, where ● Diet
it is produced through digestive and bacterial enzymatic → High purine/protein: ↑acidity (↓pH) due to sulfur-containing
action on proteins amino acids, phosphoproteins, phospholipids and nucleic
→ Highly toxic acids
1. Rapidly and efficiently removed by hepatocytes from the → Low protein: ↓acidity (↑pH) due to alkaline ash from fruits and
bloodstream vegetables
● In the liver:

BCH 5 / 6
TRANS Maynes, Meriam, Millan, Misagal CORE Mateo, Marasigan, Ng, Ocenar, Ordoñez HEAD Natural
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 C. SPECIFIC GRAVITY

Normal Specific Gravity = 1.015 – 1.025

● Represents the kidney’s capacity to form diluted or

concentrated urine
● Measures the amount of dissolved solutes or solids in the
urine

Factors Affecting Specific Gravity

● Directly proportional to: Amount of solids/solutes in the urine
● Inversely proportional to:
→ Urine volume (EXCEPT if the patient has DM)
→ Oral fluid intake
→ Diet
1. High purine diet
− ↑urine volume -> ↓specific gravity
2. Low protein diet
− ↓urine volume -> ↑specific gravity
● Specific gravity changes from 1 hour to the next

D. TITRATABLE ACIDITY
Normal TA = 150 – 500mL of 0.1N acid/day
(or 15-50 mEq H+/day)
*multiply values in mL by 0.1 to get mEq

● Formula:
→ TA (titratable acidity; mL acid/day) = (Y)(Y1)/25
→ Where:
1. Y = volume (mL) of 0.1N NaOH used
2. Y1 = volume of 24-hour urine
3. 25 = volume of urine used in test

● Kidneys help in maintaining acid-base balance by increasing or

decreasing H+ in urine
→ H+ ions secreted by the kidneys are buffered in tubular fluid
by:
1. HPO4 filtered from glomeruli
2. NH3 synthesized and secreted by renal tubular cells
● Titratable acidity of urine mainly due to acid phosphates (H2PO4
or NaH2PO4) and to a lesser extent, weak organic acids
● It employs titration of urine with a standard solution of sodium
hydroxide using phenolphthalein as an indicator
→ NaH2PO4 + NaOH -> Na2HPO4 + H2O
● Potassium oxalate is added to precipitate calcium, which might
form calcium phosphate on neutralization of urine, thus affecting
the actual endpoint

Factors Affecting Titratable Acidity

● Diet
→ High purine/protein = ↑TA
→ Low protein diet = ↓TA
● Clinical significance:
→ Metabolic acidosis: ↑TA as renal compensation
→ Metabolic alkalosis: ↓TA as renal compensation

REFERENCES
[Biochemistry] 2019/02/26. Dietary Variation Plenary. Dr. Aileen
Carlos.
2020C Trans

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