Chapter three

Biochemical assessment
Biochemical assessment means checking levels of nutrients in a blood,
urine, or stools as well as tissue and hair samples.
Biochemical tests
Biochemical tests available for assessing nutritional status can he grouped
into two general categories: static tests and functional tests.
 Static tests: are based on measurement of a nutrient or its
metabolite in the blood, urine, or body tissue. Examples are serum
measurements of albumin, calcium, or vitamin A.
 Functional tests of nutritional status are based on the idea that
the final outcome of a nutrient deficiency and its biologic importance
are not merely a measured level in a tissue or blood. Example is Dark
adaptation test (assesses vitamin A status)

The difference between plasma and serum

Serum is the liquid that remains after the clotting of blood. Whereas,
plasma is the liquid that remains when anticoagulant is added to
prevent clotting. Both contain proteins, glucose, fats, salts,
antibodies, hormones, etc…and any external substances such as
medicines.
 Stages of nutrient deficiency
1. Reduced excretion of the nutrient e.g. reduced urinary excretion
but body pool maintained.
2. Body pool smaller but no disturbance of function.
3. Biochemical signs of impaired function: reduced activity of an
enzyme or cell depletion.
4. Morphological changes and clinical signs of deficiency disease

Several factors may affect reference ranges and need to be borne in

mind when interpreting results include : age , gender , diet ,
pregnancy , time of day( hormone cortisol) ,Time of month( hormones
that affected with ministerial cycle), Time of year ( vitamin D).

The advantages and limitations of biochemical

method of nutritional assessment are:

Advantages:

 It can identify the nutritional problem in its earliest stage as

biochemical changes occur before the appearance of clinical signs
and symptoms. It is precise, accurate and reproducible.
 It is useful to correlate data obtained from other methods of nutritional
assessment such as anthropometry, clinical signs.
 Gives clear confirmed picture of the nutritional problem.
Limitations: It can be time consuming.

 It is expensive and not suitable for large scale assessment.

 It requires trained personnel, laboratory facilities.

Some important nutrients in the human body and the

laboratory tests used for estimating them:

Glucose

Blood Glucose vs. Plasma/Serum Glucose: The amount of glucose can be

measured in whole blood or in plasma or serum. The levels of glucose in
the plasma or serum are 10-15% higher than the levels of glucose in whole
blood because whole blood red blood cells contain a small amount of free
glucose, and they occupy from 40% to 45% of the blood volume

The normal range of fasting plasma glucose after fasting 8-12 hours ranges
between 70-100 mg/dL. It may rise up to 140 mg/dL two hours after eating.
If it rises above 126 mg/dL after fasting for about 12 hours, or if it rises
more than 200 mg/dL two hours after eating, this indicates having diabetes

But if the glucose level ranges from 100-125 mg/dL, it cannot be judged if
the person is normal or in a pre-diabetic stage, and followup and reanalysis
after a while is recommended

It is also possible to estimate the glucose in the urine in the laboratory or by

means of special tapes. If the level of glucose in the plasma rises above
the average tolerance of the kidneys, which is 200 mg / deciliter, glucose
appears in the urine. It is worth mentioning that glucose appears in the
urine when it is very high in the blood. If glucose appears in the urine even
though its level in the blood is normal, this indicates the presence of kidney
problems

Glycosylated hemoglobin (HbA1c) is an important tool in assessing the

extent of disease control in patients with diabetes .This test reflects the
concentration of glucose in the blood during about 120 days (3 months),
which is the lifespan of red blood cells and it expresses percentage of
hemoglobin bound to glucose from total hemoglobin. It is recommended to
perform this test every 3-6 months for diabetic patients, to assess the
status of the disease and decide whether to continue treatment or change
the treatment according to the outcome.

Protein

Protein is one of the few nutrients whose condition can be assessed using
anthropometric, biochemical, clinical and nutritional measurements.
Although each of these methods has its strengths and weaknesses,
biochemical methods are the most objective and accurate. It is preferable
to evaluate the protein status by several tests, rather than relying on only
one test.
1- Total protein measurement

Measuring the concentration of total serum proteins is an important

laboratory test in estimating protein status. These proteins are formed in
the liver from amino acids, and therefore their concentration in the
serum decreases in the case of decreased liver formation affected by
the lack of supply of amino acids. Total protein includes the sum of all
blood proteins, but the largest proportion of it is albumin and globulin

2- Albumin

Albumin is one of the most prevalent proteins in the serum and is an

important indicator of the protein status in the body and the adequacy of
protein intake. There are several points that must be taken into account
when estimating it. The first is that it is not an accurate or specialized
test to diagnose acute caloric and protein deficiency, and it is also
inaccurate to show the response to nutritional interventions. Result of a
relatively long half-life 18 to 20 days (the half-life is the time required for
a compound/component to be reduced in half) and a high amount of it (4
to 5 g/kg body weight) makes serum levels of albumin slowly respond to
nutritional change.
 3. Prealbumin

This protein is synthesized in the liver and acts as a transporter for the
thyroid hormones T3, T4 and as a transporter for the transporter protein
retinol. Because of its short half-life of about (2 to 3 days) and low body
storage (0.01 g / kg body weight), prealbumin is more sensitive to acute
changes in nutritional status and responds more quickly to changes in
the protein status than albumin or transferrin. Therefore it appears that
using it as an indicator of protein status is better than using albumin or
transferrin

4-Transferrin

Transferrin is a serum/plasma protein, formed in the liver and its function

is to bind and transport iron in the serum. Because it is stored in the
body in less amount than albumin and its half-life is shorter than the half-
life of albumin (8-9 days), it is considered a better indicator of changes in
the state of protein compared to albumin
Lipids
Lipoproteins, namely:

-Chylomicrons

-Very Low-density lipoprotein

-Low-density lipoprotein

-High-density lipoprotein

Most of the chylomicrons consist of triglycerides, while the amount of

triglycerides gradually decreases with a gradual increase in the amount
of cholesterol, phospholipids, and protein in the other three types and
the density of these groups gradually increases due to the gradual
increase in the ratio of protein to fat.

Triglycerides

Triglycerides account for about 95% of the fat stored in tissues.

Increased triglycerides by itself does not indicate a risk factor for
cardiovascular disease (CVD).
Total Cholesterol:

High levels of cholesterol are associated with atherosclerosis, a risk of

coronary artery disease.

High-density lipoprotein (HDL)

HDL is referred to as “good” cholesterol because it is believed that HDL

serves as carriers that remove cholesterol from the peripheral tissues
and transport it back to the liver for catabolism and excretion. A high
level of HDL is an indication of a healthy metabolic system.

Low-density lipoprotein (LDL)

Increased LDL levels are caused by a family history of hyperlipidemia, a

diet high in cholesterol and saturated fat, nephrotic syndrome, multiple
myeloma, diabetes, hepatic disease, and pregnancy
 Water soluble vitamins
Transketolase for thiamin status.

Glutathione reductase for riboflavin status

Aminotransferases for vitamin B6 status. In addition, the direct

measurement of plasma pyridoxal phosphate concentration is often the
preferred index nowadays

 Folate, vitamin B12, biotin, and pantothenic acid status are commonly
assessed by serum or plasma concentrations. Red cell folate is
usually considered a better index of long-term folate intakes and body
stores than plasma or serum folate

 Niacin status is usually assessed by the rate of urinary excretion of its

breakdown products N-methyl nicotinamide

 Vitamin B12 status measurement has recently been refined by

including the assay of holotranscobalamin, the most physiologically
active fraction of this vitamin in plasma and/or measuring the serum,
plasma, or urinary concentrations of methylmalonic acid, a metabolite
of fatty acid metabolism that tends to accumulate under conditions of
vitamin B12 inadequacy 27
 Methylmalonic acid, and homocysteine (a product of amino acid
metabolism which tends to accumulate under conditions of folate,
vitamin B12, or vitamin B6 deficiency), are examples of biochemical
functional indices of abnormal biochemical activity, which can help to
link micronutrient deficiencies with their functional physiological
consequences .

Vitamin C
One of the most common methods used to determine the level of
vitamin C (ascorbic acid) is its estimation in plasma or serum.
Vitamin C can also be estimated in white blood cells‖ Buffy coat
vitamin C‖, as it reflects the amount of the vitamin stored in cells and
the body in general.
Its estimation in urine is not preferred, because it is greatly affected
by the conditions of the analysis and it reflects the recent intake of the
vitamin.
Fat-soluble vitamins
Vitamin A (retinol)
Status can be classified into five categories: deficiency, borderline,
sufficient. Excess, toxic. Clinical signs appear only in cases of
deficiency and toxicity, so laboratory tests are used to evaluate the
status of vitamin A in borderline, sufficient and excessive cases.
Vitamins A and the carotenoid pigments are commonly measured in
serum or plasma. There are also less commonly used tests,
especially in the case of evaluating the vitamin A status of groups,
such as the dark adaptation test and liver biopsy. The concentration
of vitamin A in breast milk can be measured as a reference for the
vitamin A status of lactating women and to determine their response
in case of vitamin fortification.
 Plasma vitamin E
(Tocopherol) levels are usually expressed as a ratio of the vitamin
concentration in plasma to plasma cholesterol or total lipids.
One of the simplest indirect methods for determining the
concentration of vitamin E (α tocopherol) is a functional test of the
rate of erythrocyte hemolysis, based on the ability of hydrogen
peroxide (form of free radicals) to liberate hemoglobin from red cells.
If hemolysis of 10% or more occurs, this indicates a deficiency in
vitamin E. This test is considered inaccurate due to the presence of
several factors that can lead to the breakdown of red blood cells other
than vitamin E deficiency.

Vitamin D

Status is usually assessed by the concentration of 25- hydroxyvitamin

D in serum or plasma. , which is the form of the vitamin with the
highest concentration.
 It reflects the amount of vitamin D intake through food,
supplements and synthesis inside the body as a result of exposure to
sunlight.
 Vitamin K

Status is measured crudely by the rate of blood clotting, or more sensitively

and specifically by PIVKA (protein induced by vitamin K absence) and
recently by vitamin K serum or plasma levels

Iron deficiency goes through three stages

The risk of iron deficiency increases with the depletion of iron stores in the
body, and the estimation of ferritin in the serum is one of the best tests that
show the state of iron stores in the body.

 First stage - Mild deficiency

The mobilizable iron stores in the bone marrow become depleted but there
is normal production of iron-dependent proteins and a relative decrease in
the level of ferritin.

 Second stage- Marginal deficiency

Iron deficient erythropoiesis which affects iron-dependent protein

production but hemoglobin production and erythropoiesis are preserved.
Ferritin level is low.

 The third stage- Severe deficiency

Iron deficiency anemia is detected where the production of hemoglobin is

compromised and red blood cell synthesis abrogated because there is
insufficient iron for incorporation into erythroid precursor.
Tests to assess iron status

Hemoglobin

It is the iron-containing molecule in red blood cells that is responsible for

carrying oxygen and carbon dioxide.

Hematocrit

It is the percentage of red blood cells that make up blood. 3- Mean

corpuscular hemoglobin: It is the average mass of hemoglobin in one
red blood cell in a sample of blood

Transferrin

Transferrin is a glycoprotein that is produced in the liver and is present

in the blood. It is able to bind with iron and transport it to various tissues
of the body

Ferritin

It is a protein found inside cells, that controls the storage and release of
iron. Ferritin reflects the amount of iron stored in the body.
Calcium
This distribution is not constant, but it changes continuously according to the concentration of
proteins and negative ions, and according to the changes in the acidity and alkalinity of the blood.

 Serum/ plasma calcium estimation is used to help diagnose and follow

up treatment of health problems related to bone, parathyroid glands,
kidney failure, and some types of cancer. Ionized calcium is 4.5-5.6
mg/dL.

 It should be noted that the level of calcium in the serum is largely

controlled by the body, with a low correlation between the level of
calcium in the serum and the intake of calcium. Therefore, in case of
an imbalance in the level of calcium in the serum, this means that
there are other problems other than a deficiency or excess of calcium
intake.
– Hypocalcemia
– Hypercalcemia
 The normal ratio of total calcium ranges between 9-10.5 mg/dL,
while the normal ratio of free / ionized calcium is 4.5-5.6 mg/dL
 Urinary calcium: Urinary calcium levels are more responsive to
changes in dietary calcium than serum levels

 Urinary calcium increases when the volume of urine production is

high and when the kidneys' ability to reabsorb calcium is poor.
Hypocalcaemia
  The calcium level in a night urine sample can be used as an
indicator of response to calcium supplementation

Phosphorus

The level of phosphorous in serum is closely related to the

level of calcium. Hyperphosphatemia.

The normal range of phosphorus in the blood for adults is 3-4.5 mg/d

Potassium

Potassium is playing a role with other elements in maintaining the acidity

and alkalinity balance of the blood, the fluid balance in the body and the
transmission of nerve signals.

Hyperkalemia is a serious condition that leads to cardiac arrest and occurs

most often due to kidney failure, but may also result from insufficient
adrenal gland function (Addison's disease), dehydration and severe burns.

The normal range for potassium in adults is 3.5 to 5 mmol/L.

Sodium

Sodium works mainly in maintaining the acidity and alkalinity of the blood
and the fluid balance in the body

 Hyponatremia

The normal range for sodium for adults is 135-145 mmol/L.

Important note: The level of sodium in the blood does not reflect the
nutritional status of the person, but it gives an indication of the state of
fluids in the body
Zinc

 Zinc is involved in the formation of many enzymes in the body and thus
participates in many metabolic processes in the body, including protein
synthesis, wound healing, tissue growth and maintenance.

 Severe zinc deficiency is characterized by hypogonadism and dwarfism

has been observed in the Middle East.

 Although there is a test that estimates the zinc level in the plasma, its use
is inaccurate as a result of the body’s continuous maintenance of the zinc
level and its redistribution between cells. The zinc level in plasma or in
serum is also affected by non-food factors such as inflammations and
infections

Recent studies recommended that zinc status can be assessed by

combining the estimation of the zinc level in plasma and estimation of the
level of a protein that binds zinc and copper called "metalothionine".

It is found in quantities that can be estimated in serum and red blood cells,
and its quantity in cells is proportional to the status of zinc. It can be judged
that there is a deficiency of zinc in the body in case the level of both zinc
and metallothionein is lower than the normal range. It is possible to
estimate the proportion of zinc in hair and urine as well, but it is one of the
unfavourable methods for many reasons.
Iodine

 The body needs iodine in small amounts, but it is necessary for

human life and for his physical and mental development. Adult human
body contains an amount of iodine ranging between 10 to 30 mg, two
thirds of this amount in the thyroid gland, which uses iodine in the
production of “hormone thyroxine “which is necessary for many vital
functions in the body. Another portion of iodine is found in the
muscles, and trace amounts are found in the liver, ovaries, and
adrenal glands.
 Since 90% of dietary iodine is excreted in the urine, therefore, the
measurement of iodine in urine collected for 24 hours is one of the
most frequently used measurement to determine iodine intake for
several days. 13
 The iodine status can also be estimated by measuring the level of
“thyroglobulin” and the” thyroid stimulating hormone TSH” in serum,
both give an indicator of the intake of iodine for a longer period
ranging from weeks to months.
 It is worth to notice that measuring the level of thyroid hormones (T3-
T4) has no relation with the assessment of iodine status in the body.
It is worth to notice that measuring the level of thyroid hormones (T3-
T4) has no relation with the assessment of iodine status in the body.