WATER BALANCE & ELECTROLYTES

Total Body Water (42L)

Intracellular Water (28 L) Extracellular Water (14 L)
Interstitial fluid Plasma
(10.5 L) (3.5 L)

Potassium K+ Potassium
(110 mmol/L) (4 mmol/L)
K+

Na+
sodium sodium
+
(10 mmol/L) Na (135 mmol/L)

Diffusion sodium pump

The daily obligatory losses are shown as follows:

Skin 500 mL
Lungs 400 mL
Gut 100 mL
Kidneys 500 mL

T otal 1500 mL

These obligatory losses are compensated by water taken from the following sources:
Water from oxidative metabolism 400 mL
Minimum in diet 1100 mL

Total 1500 mL

ECF Osmolality
The ECF osmolality is regulated by the level of sodium and associated anions (e.g., HCO3), glucose,
urea, and proteins.

Of these solutes sodium is the major determinant of plasma osmolality.

The ECF volume is directly dependent on the sodium content and is maintained by :

1) Regulation of renal excretion of sodium or glomerular filtration rate.

About 70% of filtered sodium is reabsorbed by the earlier parts of the renal tubules.

2) Aldosterone via the RAA system.

Aldosterone
= promotes sodium reabsorption
= stimulating the release of potassium and hydrogen.
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Renin-Angiotensin-Aldosterone (RAA) System

Hyponatremia
Hypotension Angiotensinogen
Hyperkalemia

Renin (JG cells)

Angiotensin I
(lungs)
Angiotensin-converting
enzyme (ACE)

Angiotensin II Aldosterone Na retention

(adrenal cortex) K excretion

Hydration States

Dehydration
=Pure water loss or deficit.

=Water and sodium loss

Overhydration
= excessive intake of water (polydipsia)

= excessive reabsorption of water such as in cases of SIADH and ectopic

ADH secretion.

DISORDERS OF WATER METABOLISM

WATER DEFICIT
Reduced intake/water deprivation
Infancy; Elderly; & Enfeebled (Hot weather complicates)
Defective thirst
Head injuries; and postneurosurgery
Excess solute intake
Formula feeding to supplement nutrition; NGT with heavy solute
Sweating
Renal loss of water
Primary due to loss of renal concentrating ability in nephropathy;
Secondary due to diabetes mellitus; & Osmotic dieresis

WATER EXCESS
Compulsive water drinking
Increased intake with inappropriate secretion of ADH
Renal abnormality that limits or prevents water excretion
Congestive heart failure (CHF) (underperfusion of kidneys)
Cirrhosis of the liver with ascites
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Function of Electrolytes
Maintenance of osmotic pressure and hydration e.g., Na+

Buffering functions e.g., HCO3-

Activators in enzyme reactions e.g., Mg++

Normal neuromuscular excitability e.g., Ca++

Redox reaction (electron transport) e.g., Fe++/Fe+++

Levels of Electrolytes in the Plasma (mmol/L)

Plasma Interstitial Intracellular

Fluid
Cations
Na+ 142 140 10
K+ 5 4.1 150
Ca++ 5 4.1 -
Mg++ 5 3.0 40
Total 155
Anions
Cl- 103 115 15
HCO3- 27 27 10
PO4 3- 2 2 100
SO4 2- 1 1.1 20
Organic Acids 6 3.4 -
Proteins 16 10 60
Total 155

Anion Gap

The anion gap refers to the difference between the sums of the concentrations of the principal
cations (e.g., Na+ and K+) and of the principal anions (e.g., CI -and HCO3-).

It represents the unmeasured net negative charge on plasma proteins.

Methods of calculating Anion Gap. The anion gap (AG) may be measured by any of the following
formula:
1. AG = Na+ - (Cl- + HCO3 -) NV: 7-14 mmol/L
2. AG = (Na+ + K+) - (Cl- + HCO3-) NV: 10-18 mmol/L

ALTERATIONS IN ANION GAP

INCREASED ANION GAP
Chloride Normal; Normal or low bicarbonate (increased anions)
Diabetic ketoacidosis
Uremic acidosis due to sulfates, phosphates and fixed acids; Starvation
Alcoholic ketosis (ethanol metabolites, lactate)
Lactic acidosis due to hypoxia/hypoperfusion
Exogenous poisons (ketones, lactate, salicylates, alcohols)
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NORMAL ANION GAP ACIDOSIS
Hyperchloremic acidosis
Diarrhea
RTA
Hyperalimentation
Early renal failure

DECREASED ANION GAP (<6 mEq/L)

Cationic myeloma proteins
Hyperlipidemia with decreased plasma content of water & all electrolytes
Erroneous report

Sodium
= most abundant cation in the extracellular fluid.

= accounts for about 92% of the osmotically active solutes in the plasma. Its amount also determines
the ECF volume.

= 70% of sodium is freely exchangeable while 30% is complexed in the bone.

Regulators of Sodium Level

Diet
Kidney
= renal threshold for sodium is 110-130 mmol/L.
= about 70-80% of filtered sodium is reabsorbed at the proximal tubule
RAA
= aldosterone promotes retention of sodium in exchange of secretion of K+ & H+ ions.
Atrial natriuretic factor (ANF)
= this cardiac peptide promotes natriuresis (urinary excretion of sodium) and relaxation of the
vascular smooth muscle (vasodilatation)

Reference Ranges
sodium in the extracellular fluid is 135-145 mmol/L
in the intracellular fluid, it is within 4-10 mmol/L

Determination of Sodium
Serum sodium may be measured using emission flame photometry (EFP), atomic
absorption spectrophotometry (AAS), or ion-selective electrodes (ISE).

Colorimetric method called Albanese-Lein

= involves combining sodium with zinc uranyl acetate to produce sodium uranyl acetate
precipitate. Addition of water to the precipitate produces a yellow solution

ABNORMALITIES OF SODIUM AND THE EXT RACELLULAR FLUID (ECF)

HYPONATREMIA
Total-body Na & ECF volume LOW
GIT fluid loss
Burns
“Third compartment” accumulation (ascites, ileus)
Salt-losing renal disorders
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Diuretic overuse
Total-body Na & ECF volume NORMAL
Acute water intoxication, usually iatrogenic
Syndrome of inappropriate secretion of ADH (SIADH)
Glucocorticoid deficiency
Severe whole-body K depletion
Total-body Na & ECF INCREASED
Acute renal failure with superimposed water load
CHF
Cirrhosis
Nephrotic syndrome

HYPERNATREMIA
Total-body Na NORMAL & ECF volume LOW
Excessive insensible loss: fever, thyrotoxicosis
Diabetes insipidus
Total-body Na & ECF volume LOW
Gastroenteritis
Osmotic diuresis
Pronounced sweating
Total-body Na INCREASED proportionately more than INCREASED ECF volume
Salt ingestion, deliberate or accidental
Inappropriate IV therapy

HYPEROSMOLALITY, without Na alterations

High blood ethanol (high osmolality but plasma not
hypertonic)
Hyperglycemia
Radiographic contrast media

Potassium
Potassium is the major intracellular cation.

It is about 20 times greater in concentration inside the cells than outside.

= about 90% of potassium is free or exchangeable
= only 10 % is bound in the red blood cells, bone and brain tissues.

Normal values of potassium in serum samples are in the range of 3.8-5.0 mmol/L.
= elevated levels of potassium (>7.5 mmol/L)can seriously inhibit the irritability of muscles,
including the heart and may lead to paralysis or cessation of heartbeat
= low serum potassium (<3.0 mmol/L) may increase muscle irritability and cause
heartbeat during systole.

Measurement of Potassium
Like sodium, potassium may be measured by flame photometry, atomic absorption
spectrophotometry and ion-selective electrode.

Colorimetric procedure for potassium is Lockhead and Purcell that uses potassium cobaltinitrite. A
blue sodium potassium cobaltinitrite is produced with addition of phenol reagent.

Sodium and Potassium Assay Notes

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=anticoagulants which tend to increase plasma volume e.g., oxalates, lowers sodium levels.
= blood samples taken after physical exercise or muscular activity have lower sodium.
= water used in the assay must be free of electrolyte traces and the thumb must not be used when
mi xing the tubes since the skin may contain sodium chloride.
= serum potassium levels are usually higher than those obtained using plasma samples due to
platelets release potassium during the clotting process
= among the causes of spuriously high potassium are:
1. Increased platelet count
2. Prolonged application of tourniquet due to juxtavenular cellular injury production leakage of
potassium
3. Increased muscular activity e.g., repeated or excessive clenching of fist prior to and during
drawing of blood
4. Hemolyzed specimen (greatest error)
5. Contamination with potassium EDTA

ABNORMALITIES OF SERUM and WHOLE-BODY POTASSIUM

HYPERKALEMIA
Inappropriate cellular metabolism
Insulin deficiency; Acidemia; Hypoaldosteronism; & Cell necrosis (burns, crush, hemolysis,
antileukemia therapy)
Decreased renal excretion
Acute renal failure; Chronic interstitial nephritis; Tubular unresponsiveness to aldosterone; &
Hypoaldosteronism
Increased K intake
Inappropriate use of salt substitutes for K+ replacement
Potassium salts of antibiotics

HYPOKALEMIA
Inappropriate cellular metabolism
Alkalemia; Familial periodic paralysis; & Very rapid generation of cells (leukemia, treated
megaloblastic anemia)
Increased excretion
Vomiting and/or diarrhea; Diuretic overuse; Hyperaldosteronism; & Renal tubular acidosis
(RTA)
Decreased K intake
Anorexia nervosa; Diet deficient in vegetables and meats; & Clay eating (binds K and prevents
absorption)

Chloride

Chloride is the major extracellular anion.

It is for the maintenance of electrolytes balance, hydration, and maintenance of osmotic pressure.

Measurement of Chloride
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Chloride may be measured by the ISE method
= membrane of the chloride ISE is a composite of silver sulfide and silver chloride
= only bromide can possibly cause interference.

Coulometric-amperometric method with adaptation of this is the Cotlove titrator or chloridometer.

Zall color reaction has been used in some semi-automated chloride analyzers
= reagent contains mercuric thiocyanate and ferric nitrate
= the chloride ions displace the thiocyanate ions to form soluble but undissociated mercuric
chloride
= releasing in the process an equivalent amount of thiocyanate
= this reacts with ferric ions derived from the ferric nitrate to produce reddish brown ferric
thiocyanate

Mercuric nitrate titration method of Schales and Schales

= the mercuric nitrate forms soluble but virtually undissociated mercuric chloride, which does
not affect the indicator diphenylcarbazone
= as soon as all the chloride ions are thus combined, the next drop of mercuric nitrate added will
cause the indicator to change to from colorless or faint pink to violet
= the volume of mercuric nitrate required to produce the endpoint is a measure of the amount of
chloride present

CSF Chloride
= normally higher than that of serum because the protein concentration in cerebrospinal fluid is
low, hence, there are practically no proteinate anions.
= about 115-132 mmol/L
= falls to about serum levels in cases of bacterial meningitis when protein concentration in the
CSF is elevated

Sweat Chloride
= screens for cystic fibrosis
= about 50 mg sweat is needed to test for chloride
= sweat-inducing drug, pilocarpine, is introduced into a limited area of the skin by means of
an electric current flowing between two electrodes attached to a limb (of a child) or the
back (on an infant), technique is called iontophoresis
= normal sweat chloride is about 5-40 mmol/L and most patients with cystic fibrosis have levels
above 60 mmol/L.

HYPERCHLOREMIC ACIDOSES

Renal Tubular Failure

Defective exchange of H+ for Na+ causes increased urinary excretion of
HCO3-
Urine often inappropriately alkaline
Urine: Na+ increased, K+ increased, Cl- decreased
Ca++ excretion and bone demineralization, if prolonged
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Gastrointestinal Loss of HCO3-

Na+ & K+ loss in intestinal fluid
Fluid and electrolyte loss  hypovolemia
Increased BUN and creatinine, from dehydration and decreased GFR
Urine Na+ and Cl- very low

Calcium

Calcium is the most abundant cation in the body. It is 90% bound to the skeleton. In the bone. Its
combines with phosphates to form the hydroxyapatite crystals which provide strength to the
bone.
Functions of calcium
Structural Bone
Neuromuscular Control of excitability
Release of neurotransmitters
Initiation of muscle contraction
Catalytic Coenzyme for coagulation factors
Signal transduction Intracellular secondary messenger

Calcium Regulation
Parathyroid Hormone (PTH)
= increases blood calcium levels
= secretion is inhibited by hypercalcemia and calcitriol
Target PTH Action Effect
Organ
Rapid release of calcium from Increased plasma Ca
Bone osteoclastic resorption
Increased Ca reabsorption Increased plasma Ca

Decreased Pi reabsorption Decreased plasma Pi

Kidney
Increased 1-hydroxylation of 25- Increased Ca & Pi
Ohcholecalciferol absorption in the gut

Decreased bicarbonate reabsorption Acidosis

Calcitriol or Activated Vitamin D3
= also known as 1.25-didydroxy-cholecalciferol
= it is stimulated by increased PTH and decreased phosphate
Calcitonin
= produced by the C cells of the thyroid gland
= decreases blood calcium level (hypocalcemic hormone)
Measurement of Calcium
Aside from ISE (reference method), atomic absorption spectrophotometry and flame
photometry.

Three Measurement Approaches

1) Precipitation of calcium as an insoluble compound followed by titration or
colorimetric methods
ammonium oxalate (Clark-Collip)
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chloranilic acid (Ferro-Ham method)
picrolonic acid
2) Formation of colored complexes calcium and a variety of dyes followed by
colorimetric determination of the complex
Alizarin
O-cresolphthalein complexone (Ca-OCP)
Calcein (Diehl-Ellingboe method)
Ammonium purpurate (Murexide)
Nuclear fast red
3) Removal of calcium from a colored complex by titration with a chelating agent
EDTA (ethylene diamine tetraacetic acid)
EGTA (ethylene glyco-bis(2-aminoethyl ether)- tetraacetic acid)
Dyes
The endpoint is reached by recovery of the original color of the dye or the
disappearance of the fluorescence of the calcium-dye complex.

O-cresolphthalein complexone method

= the dye binds calcium tightly in alkaline solution to form a highly colored complex with an
absorbance at 578 nm
= the reaction mixture contains 8-hydroxyquinoline to bind magnesium and prevent its
interference,
= urea to decrease the turbidity of a lipemic serum and increase color intensity of the calcium-
dye complex, and
= ethanol to decrease the absorbance of the blank

Normal serum total calcium (Cat) falls within the range of 8.5 to 10.4 mg/dL (2.13 to 2.60 mmol/L).
Ionized calcium (Ca2+) in plasma, serum or whole blood is within the normal range of 4.68-5.32
mg/dL (1.17 to 1.33 mmol/L).
Multiply mg/dL by 0.25 to convert to mmol/L

Inorganic Phosphate
= in the body exists only as inorganic phosphate esters
= about 80% of the phosphates are incorporated into the bone together with calcium
= most organic phosphates are present inside the cells as components of molecules e.g., the DNA ,
phospholipids, ATP, etc.
= in contrast, most inorganic phosphates are mostly confined in the extracellular fluid where they act
a s b u f fe r s
= excreted principally via the urine
= phosphate homeostasis is closely linked with calcium regulation since the same hormones regulate
the levels of the two minerals
PTH, for example, stimulates the kidney to excrete phosphate while conserving calcium

Usually, the relationship between calcium and phosphorus is inverse.

Measurement of Phosphates
Fiske-Subbarow method
= protein-free filtrate is prepared using trichloroacetic acid
= conversion of the inorganic phosphate in the sample to the heteromolybdenum blue by a
reaction with ammonium molybdate and the reducing agent, aminonaphthol sulfonic acid (pictol)
= the absorbance of the complex is measured at 700 nm

Daly-Ertingshausen method
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= the inorganic phosphates is converted into phosphomolybdate polyacid by a reaction with
ammonium molybdate in sulfuric acid
= precipitation of proteins is prevented using a wetting agent called Tween 80
= OD of the phosphomolybdic acid is measured at 340 nm

Magnesium

= the 4th most abundant cation in the body

= majority of this mineral is stored in the bones in complex with calcium and phosphate
= about 70% of magnesium is free and only 30% is bound to protein
= most of the magnesium in the body is located within the cell.
= an essential activator of several enzymes e.g., phosphateses, kinases, phosphorylases and
enolases.
= also necessary in the oxidative phosphorylation occurring in the mitochondria.
= therapeutic agent and has an anti-convulsant laxative and antacid effects

Measurement of Magnesium
Magnesium may be measured by ISE, AAS, colorimetric or fluorometric analysis.

Calmagite (3-hydroxy-4 [(6-hydroxy-m-toly)azo]-1-naphthalene-O-sulfonic acid)

= in the presence of polyvinylpyrrolidone (used to minimize the effects of serum proteins), a
violet complex forms which absorbs light at 520 nm
= the reagent used contains amphoteric betaine detergent Empigen BB to shift the wavelength
of the blank, strontium chelate to mask the effect of calcium, and triethanolamine to mask
the effects of iron

Methylthymol blue
= complex formed is measured at 510 nm
=used in the DuPont aca analyzer.

Titan yellow
= method is called Dye-Lake method
= magnesium reacts with an alkaline solution of titan yellow in the presence of
polyvinylpyrrolidone to form a red lake colloidal precipitate

Fluorometric analysis include the use of the either hydroxyquinoline or calcein.

Normal values magnesium fall within the range of 1.3 to 2.1 mEq/L or 0.65 to 1.05 mmol/L.

TRACE ELEMENTS

Trace elements are present in the body in very small amounts usually less than 1 microgram
per gram of tissue. They form part of the micronutrients of the body and can be subdivided into four
(4) major groupings based on their physiological function:
1) Essential elements for normal growth, development and maintenance with established
Recommended Daily Allowance (RDA) (e.g. Fe, Zn, I, and Se)
2) Elements with definite role in the body but with no RDA yet established (e.g. Cu, Mn, Cr,
Co, Mo, & F)
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3) Elements found consistently in body tissues in ultratrace amounts and not known to have
a definite role or detriment to the body (e.g. Li, Ni, Sn, Si, and V)
4) Elements with no known function in the body and cause pathological changes/toxic if
present (e.g. Al, Be, Cd, Hg, Pb, and As)

Dietary Reference Intakes (DRI) Definitions

(According to the Food and National Board of the National Academy of Sciences in 1997)

Recommended Dietary Allowance (RDA) is the average daily dietary intake level that is
sufficient to meet the nutrient requirements of nearly all (97 to 98%) healthy individuals in a
particular life stage and gender group.

Adequate Intake (AI) is a recommended intake value based on observed or experimentally

determined approximations or estimates of nutrient intake by a group or groups of healthy
individuals, which is assumed to be adequate; it is used when an RDA cannot be
determined.

Tolerable Upper Intake Level (UL) is the highest level of daily nutrient intake that is likely
to pose no risk of adverse health effects for almost all individuals in the general population.
As intake increases above the UL, the potential risk of adverse effects increases.

Estimated Average Requirement (EAR) is a daily nutrient intake value that is estimated
to meet the requirements of half of the healthy individuals in alife stage and gender group;
used to assess dietary adequacy and serves as the basis for RDA.

Copper

In the blood, copper is seen in red blood cells or is bound to transport proteins e.g., albumin, and
ceruloplasmin.

Ceruloplasmin is necessary for the absorption of iron to the ferric state, a prerequisite for binding by
transferrin. It has a peroxidase activity.

Copper is important in erythropoiesis (hemoglobin synthesis) and catalytic activity of several enzymes
e.g., cytochrome oxidase and uricase.

Serum copper may be measured by AAS.

To convert to SI, multiply ug/dL by 0.157 to get values in umol/L.

Iron

Total body iron in humans is approximately 3-5 g with about 70% incorporated in the red blood
cells, and about 25% is found in the reticuloendothelial system, incorporated with ferritin
and hemosiderin as stored iron.

Two forms of iron in the body are:

Ferrous iron = found in oxyhemoglobin and reduced hemoglobin
Ferric iron = found in ferritin, hemosiderin, transferrin and methemoglobin
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The two other proteins that are involved in the transport of iron are:
Haptoglobin. This binds hemoglobin and services to facilitate disposal of the iron from this
molecule
Hemopexin. This binds heme to avoid to aid its removal from the circulation

Measurement of Iron
Ferrozine method
= where serum proteins are precipitated in an acid solution containing thioglycolic acid that
reduces ferric to ferrous ion, thereby dissociating the iron from its binding to transferrin
= chromogen ferrozine is then added to the supernate to form a highly colored ferrous complex
which is measured at 562 nm.

Normal serum iron concentration falls within 65 to 165 ug/dL (11.6-29.5 umol/L) for men and 45 to
160 ug/fL (8.1-28.6 umol/L) for women.

Higher values are obtained in the morning due to diurnal variations.

Total Iron Binding Capacity (TIBC) and Transferrin Saturation

A known amount of ferric ions, more than sufficient to fully saturate the serum transferrin with
iron, is added to a serum sample. The excess ferric ions, not bound to transferrin, is removed by
addition of a small amount of buffered ion-exchange resin.

The sample is diluted and centrifuged, and an aliqout of the supernate is analyzed for iron
content of the fully saturated transferrin - this value is the TIBC.

The % saturation of transferrin is measured as followed.

Transferrin Saturation = Serum Fe x 100

(% saturation) TIBC

TIBC varies from 260 to 440 ug/dL (46.5-78.8 umol/L). Transferrin saturation ranges from 20-
50%.

Zinc Protopophyrin/Heme Ratio (ZPP/H)

= excellent screening test for detecting iron deficiency anemia and for monitoring the course of
therapy.
= hemoglobin in a drop of blood is converted to cyanmethemoglobin by treatment with cyanide
containing reagent
= portion of the mixture is placed on a cover slip and introduced into a ProtoFlour
hematofluorometer which measure simultaneously the light absorbed by the film of
cyanmethemoglobin at 424 nm and the fluorescent light emitted at 595 nm by zinc
protoporphyrin.
= the results are displayed as a ratio of umol ZPP/mol heme.

The normal ratio is 30 to 80 umol ZPP/mol heme.

= normal in thalassemia but abnormal in iron deficiency anemia.

= the ratio is elevated in all types of iron deficiency syndromes and chronic exposure to lead.
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BIOLOGICAL ROLES OF ESSENT IAL TRACE ELEMENTS & ASSOCIATED ABNORMALITIES

Element Biological Role Comments Deficiency/Abnormality/ Toxicity
Chromium Glucose metabolism Potentiates insulin Glucose intolerance in deficiency
action
Cobalt Component of vitamin No other function Pernicious anemia
B1 2 known in man
Copper Co-factor for oxidase 90-95% Cu bound to Inherited diseases: W ilson’s & Menke’s
enzymes ceruloplasmin
Fluorine Inhibits dental caries; Usually supplied as Excessive intake cause fluorosis
therapeutically improves supplement to drinking
hydroxyapatite crystal water
quality in bones
Iodine Component of T3 & T4 Concentrated in the Iodine deficiency still occurs in various
thyroid; geographic areas
supplementation by
addition to salt is
common
Iron Component of heme In plasma, bound to Hypochromic, microcytic anemia in
enzymes; hemoglobin, transferrin, stored as deficiency
cytochromes ferritin
Manganese Required for Component of Deficiency not known in man
glycoprotein and mitochondrial peroxide
proteoglycan syntheses dismutase
Molybdenum Component of sulfite Essential fo r Deficiency reported in total parenteral
and xanthine oxidases production of uric acid nutrition (TPN) patient; inability to
metabolize methionine
Selenium Component of Antioxidant properties, Deficiency may occur where soil Se is
glutathione peroxidase Se and vitamin E act low and in long-term TPN patients with
& iodinothyronine-5’- synergistically inadequate supplements
deiodinase
Silicon Involved in calcification Role in bone, cartilage, Deficiency: Impairment of normal growth
of bones and connective tissue in animals; silicosis may occur from
is poorly understood industrial exposure
Zinc Co-factor or component Involved in many Deficiency: Growth failure,
of more than 200 metabolic processes; hypogonadism, impaired wound healing;
metalloenzymes protein synthesis;
immunological Genetic disease: acrodermatitis
function; growth & enteropathica-impaired absorption;
development
Toxicity:vomiting, gastrointestinal
irritation
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MEASUREMENT MET HODS & FOOD SOURCES OF TRACE ELEMENTS

Trace Element Methods Food Sources
Chromium Graphite Furnace AAS with Brewer’s yeast, mushrooms, molasses, nuts,
Zeeman background correction wine, beer, asparagus, prunes, meats,
cheeses, and whole grains
Copper Graphite furnace AAS with Shellfish, liver, kidney, egg yolk, and some
Zeeman background correction legumes
Fluorine ISE Natural or artificially supplemented drinking
water
Iodine ISE for iodide levels and Marine fish and seaweeds; iodized salt
immunoassay for thyroid products
hormones
Manganese Graphite furnace AAS with Bran flakes and wheat products
Zeeman background correction;
Magnesium nitrate as matrix
modifier
Molybdenum Graphite furnace AAS with Milk, milk products, organ meats, and dried
Zeeman background correction legumes and some cereals
Selenium Graphite furnace AAS with Organ meats, seafoods, cereals and grains,
Zeeman background correction; dairy products, and fruits and vegetables
Nickel nitrate or reduced grown in seleniferous areas
palladium as matrix modifier
Zinc Flame AAS for serum, plasma, Seafoods, meats, milk, and eggs; low in
urine, and for erythrocytes fibrous plants and vegetables

ACID-BASE BALANCE

Blood gas analysis routinely involves analysis of blood gases oxygen and carbon dioxide, and blood
pH.

The preferred sample if arterial blood collected in heparinized tubes.

The normal arterial pH falls within the range of 7.35 – 7.45 (average of pH 7.4). This is equivalent to
a molar hydrogen ion concentration of 4.5 x 10-8M to 3.5 x 10-8M buffer.

The bicarbonate buffer system is illustrated as follows:

H2CO3  H+ + HCO3- Equation 1

H2CO3 and HCO3 act as the conjugate acid-base pair with the latter acting as the base. The Ka
(acidity constant) for the equation can be written as follows:

Ka = [H+][HCO3-]
[H2CO3] Equation 2

where the brackets represent the molar concentration. The equation tells that the higher the
hydrogen ion concentration, the higher is the acidity constant.
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If the hydrogen ion concentration is to be solved then the equation becomes

[H+] = Ka [H2CO3]
[HCO3-] Equation 3

Since pH = –log [H+], th e n

pH = -log Ka – log [H2CO3] Equation 4

[HCO3- ]

Or ,
pH = [HCO3- ]_____
pKa + log [H2CO3] Equation 5

pH = pKa + log [HCO3- ]

[H2CO3]

T his is called the Henderson-Hasselbalch equation.

The pKa is a constant and it depends on the buffer involved.

The pKa is the pH at which the molar concentration of the acid is equal to the molar concentration of
its conjugate base.

It is at this pH where the system exerts its maxi mum buffering activity. Usually the range of pH at
which a buffer is effective is within pKa + 1 pH unit.

The bicarbonate buffer has a pKa of 6.1 which means that it is effective in maintaining the pH of a
solution within the range of pH 5.1 to pH 7.1.

CAH CAH
CO2 + H2O ----- H2CO3 ------- H+ + HCO3- Equation 6

In the peripheral tissues

= the bicarbonate diffuses out of the red blood cell, to maintain electrical neutrality, this
diffusion of bicarbonate is accompanied by as shift of chloride into the red blood cells
(chloride shift) which is mediated by a transport protein located in membrane of the red blood
cell.

In addition to the respiratory component of the acid-base balance, the levels of bicarbonates in the
blood is also closely regulated by the kidney.

= bicarbonates are readily filtered in the glomerulus but it is absorbed in the proximal tubule
especially when a lot of the base needed e.g., in cases of acidosis.
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= this mechanism forms the metabolic component of the acid-base balance.
In summary, acid-base balance is controlled by chemical buffers primarily bicarbonate, the lungs and
the kidney. It can be represented as follows:

pH = [HCO3-] which is a function of the kidney (metabolic component)

[H2CO3] which is a function of the lungs (respiratory component)

Normally, the levels of bicarbonate and carbonic acid are maintained at a ratio of 20:1.

Total CO2

Total CO2 consists of the HCO3 , undissociated H2CO3, dissolved CO2, and carbamino-bound CO2.

The bicarbonate is by far the largest (~95% of the total) and accounts for all but approxi mately 2
mmol/L of the CO2 content.

The carbamino fraction is negligible in serum, but is appreciable in whole blood because of the
presence of hemoglobin.

Generally, the CO2 content is measured by automated methods or using a CO2 electrode.

Routinely, the total CO2 content is assumed to be equal to the sum of the dissolved CO2 and
bicarbonate.

This can be expressed in mmol/L. The normal value of total CO2 is 23 – 27 mmol/L.

pCO2 or dissolved CO2

= constitutes about 5 – 10% of the total CO2 content usually expressed in mmHg

= the normal value of 35 – 45 mmHg.

Sample Problem.
Given a pCO2 of 44 mmHg and total CO2 of 29 mmol/L. Solve for the pH.

= first, convert pCO2 in mmHg to dissolved CO2 by multiplying the solubility coefficient of CO2
gas which is a constant (0.03 mmol/L/mmHg) i.e.,
44 mmHg x 0.03 mmol/L/mmHg = 1.32 mmol/L

= then determine the HCO3 concentration by finding the difference between total CO2 and
dissolved CO2 concentration
29 mmol/l – 1.32 mmol/L = 27.68 mmol/L

= Calculate the pH as follows:

pH = 6.1 + log [HCO3- ] = 6.1 + log [HCO3-]
[H2CO3] [pCO2]

pH = 6.1 + log 27.68 mmol/L

1.32 mmol/L = 7 .4 2
 17
Physiologic Buffers

Bicarbonate Buffer.

Hemoglobin.
= this is a major buffer localized inside the red blood cells.
= in the peripheral tissues, carbon dioxide accumulates as a waste product of metabolism.
= as the pressure of carbon dioxide increases in the plasma, the gas diffuses into the red blood
cells where it reacts with water to form carbonic acid as catalyzed by carbonic
anhydrase (also known as carbonic dehydratase).
= the carbonic acid readily splits into hydrogen ions and bicarbonate.
= the hydrogen ion combines with hemoglobin which then releases the oxygen for the tissues.
Hemoglobin therefore can also act as buffer.

Phosphate Buffer.
This buffer system has a minor role in the blood. Instead, along with plasma proteins containing
especially the amino acid glutamine, it is important for the excretion of hydrogen ions in the
kidney. The conjugate acid-base pair of this buffer is shown as follows:
H2PO4  H+ + HPO42-
The pKa of this reaction is 6.8.

Plasma Proteins.
The amino acids present in the proteins are amphoteric and they can act as buffer.

Transport of Blood Gases

The transport of oxygen and carbon dioxide back and forth the lungs and the peripheral tissues is a
function of hemoglobin in red blood cells. Hemoglobin exists in two forms:

“T” or taut structure (deoxyhemoglobin) which has a low affinity for oxygen.
“R” or relaxed structure (oxyhemoglobin) which has high affinity for oxygen.

Interstitial Fluid
H2O + CO2  H2CO3 Renal Tubular Cell

Na+
- +
HCO3 H NH3

HPO42- HPO42- + H+ Na+ NH3

Renal Tubular
H2PO42- Lumen

NH4+

Renal hydrogen ion excretion by the phosphate and protein buffers.

 18
In the peripheral tissues where the oxygen tension is very low because oxygen is utilized during
metabolism, the hemoglobin molecule exists in the taut form in order to prevent uptake of the
delivered oxygen.

In contrast, the relaxed form is favored in the lungs where oxygen tension is very high. This allows
uptake of oxygen by the hemoglobin molecule.

The affinity of hemoglobin with oxygen depends on many factors. This can be shown with the
oxygen-hemoglobin dissociation curve.

Decreased
100 PCO2
Temp
2,3-BPG Lungs
Shift to the Right
Increased
% saturation of pH Increased
PCO2
hemoglobin
Temp
50 Shift to the 2,3-BPG
Left
Decreased
Peripheral pH
Tissues

40
Partial pressure of oxygen (pO2) in mmHg

The Oxygen-Hemoglobin Dissociation Curve

= the curve is sigmoid in shape
= exhibits cooperative effects, in that, at lower oxygen tension, the affinity of hemoglobin with
oxygen is very small, however, once the hemoglobin molecule has started binding with
oxygen, its affinity for the succeeding oxygen molecules increases

Comparison of Arterial and Venous Blood

Parameter Arterial Blood Venous Blood
Chloride Higher Lower
Bicarbonate Lower Higher
PCO2 Lower Higher
PO2 Higher Lower
pH Less More

Blood Gas Assay Notes

Arterial blood is required for pO2 measurement.

Venous blood may be taken for pH and pCO2 if it is drawn without stasis (no tourniquet) and without
the patient clenching the fist.
 19
“Arterialized” venous blood may be obtained by heating the hand and forearm in water at 45oc for 5
minutes and then drawing blood from the dilated veins on the back of the hand.

W hen the blood sample is left in open air, carbon dioxide diffuses from the blood to the surrounding
air, reducing the pCO2 in the blood thereby increasing the pH.

In contrast, oxygen diffuses from the air into the blood since pO2 in air is greater than that in whole
blood.

Measurements of blood pH, pCO2 and pO2 may be done simultaneously in a blood gas instrument.
The pH is measured by a micro glass electrode.

pCO2 is measured by a Severinghaus electrode while pO2 is measured by the Clark electrode.

Natelson-Van Slyke method is an example of a gasometric method for carbon dioxide.

Disorders of Hydrogen Ion Homeostasis/Acid-Base Disturbances

W hen the bicarbonate level is primarily defective, the condition is referred to as metabolic in nature.

If the level of carbonic acid is primarily defective, the condition is classified as respiratory in nature.

Metabolic Acidosis.
= bicarbonate is very low resulting in a low pH

= can be compensated for by the lungs by hyperventilation lowers the carbonic acid level
restoring the pH.

= clinical observations include:

Kussmaul respirations; Shock, coma, moderate hypokalemia

Metabolic Alkalosis.
= bicarbonate is very high resulting in high pH

= can be compensated for by the lungs hypoventilation which increases carbon dioxide in the
blood.

= clinical observations include:

Paresthesias; tetany; hypokalemia, & weakness

Respiratory Acidosis.
= seen when the carbonic acid levels are very high.

= can be compensated for by the kidneys by reabsorb a lot of bicarbonates to restore the pH of
the blood.

= clinical observations include:

ACUTE: Air hunger, disorientation
CHRONIC: Hypoventilation, hypoxemia, cyanosis
 20
Respiratory Alkalosis.
= occurs when the level of carbonic acid is very low.

= compensated for by the kidneys by allowing more excretion of bicarbonates in the kidney

= clinical observations include:

ACUTE: Hyperventilation, paresthesias, light-headedness
CHRONIC: Hyperventilation, latent tetany
 21
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