BIOCHEMICAL PRINCIPLES OF NUTRITION

biochemical principles of nutrition

 

Wheher a man eats for living or lives for eating, food is his prime concern. Nutrition may be defined as the utilization of food by living organisms. Biochemists have largely contributed to the science of nutrition.

Nutrition   significantly   promotes   man's development, his health and welfare. The subject nutrition, perhaps, is the most controversial. This is due to the fact that nutrition is concerned with food and everyone feels competent enough to talk like an expert on nutrition. Further, high public awareness and the controversial reports by scientists also contribute to the controversy.

Methodology in nutrition : Most of the existing knowledge on nutrition is originally derived from animal experimentation. This is despite the fact that there may exist several differences in the biochemical composition between man and animals! For instance, some animals can synthesize ascorbic acid while man cannot do so.

Study of human nutrition : The study of nutrition may be logically divided into three areas—ideal nutrition, undernutrition and overnutrition. Ideal nutrition is the concern of everyone. Undernutrition is the prime concern of developing countries while overnutrition is a serious concern of developed countries.

NUTRITION AND ENERGY SUPPLY

Food is the fuel source of the body. The ingested food undergoes metabolism to liberate energy required for the vital activities of the body.

Energy content of foods

The calorific value (energy content) of a food is calculated from the heat released by the total combustion of food in a calorimeter.

Unit of heat : Calorie is the unit of heat. One calorie represents the amount of heat required to rise the temperature of one gram of water by 1 °C (i.e. from 15° to 16°C). A calorie is too small a unit. Therefore, it is more conveniently expressed as kilocalories (1,000 times calorie) which is represented by kcal or simply Cal.

The joule is also a unit of energy used in some countries. The relationship between calories and joules (J) is: 1 Cal (1 kcal) = 4,128 KJ.

Calorie value of foods : The energy values of the three principal foodstuffs –carbohydrate, fat and protein—measured in a bomb calorimeter and in the body are given in the Table. The carbohydrates and fats are completely oxidized (to CO₂ and H₂O) in the body; hence their fuel values, measured in the bomb calorimeter or in the body, are almost the same. Proteins, however, are not completely burnt in the body as they are converted to products such as urea, creatinine and ammonia, and excreted. Due to this reason, calorific value of protein in the body is less than that obtained in a bomb  calorimeter.  The  energy  values  of carbohydrates, fats and proteins (when utilized in the body) respectively, are 4, 9 and 4 Cal/g.

It must be noted that the nutrients, namely vitamins and minerals, have no calorific value, although they are involved in several important body functions, including the generation of energy from carbohydrates, fats and proteins.

Respiratory quotient of foodstuffs

The respiratory quotient (R. Q.) is the ratio of the volume of CO₂; produced to the volume of O₂ utilized in the oxidation of foodstuffs.

 

 

Mixed diet : The R. Q. of the diet consumed is dependent  of the  relative  composition  of carbohydrates, fats and proteins. For a normally ingested diet, it is around 0.8.

UTILIZATION OF ENERGY IN MAN

Man consumes energy to meet the fuel demands of the three ongoing processess in the body.

1. Basal metabolic rate

2. Specific dynamic action

3. Physical activity.

Besides the above three, additional energy supply is needed during growth, pregnancy and lactation.

BASAL METABOLIC RATE

Basal metabolism or basal metabolic rate (BMR) is defined as the minimum amount of energy required by the body to maintain life at complete physical and mental rest in the post-absorptive state (i.e. 12 hours after the last meal).

Under the basal conditions, although the body appears to be at total rest, several functions within the body continuously occur. These include working ot heart and other organs, conduction ot nerve impulse, reabsorption by renal tubules, gastrointestinal motility and ion transport across membranes (Na⁺-K⁺ pump consumes about 50 % of basal energy).

Measurement of BMR

Preparation  of the subject  :  For the measurement of BMR the subject should be awake, at complete physical and mental rest, in a post-absorptive state and in a comfortable surrounding (at 25°C).

Measurement : The BMR is determined either bv the apparatus of Benedict and Roth (closed circuit device) or by the Douglas bag method (open circuit device). The fomer is more frequently used.

By Benedict-Roth method, the volume of O₂ consumed (recorded on a graph paper) by the subject for a period of 2-6 minutes under basal conditions is determined. Let this be A liters for 6 minutes. The standard calorific value for one liter O₂ consumed is 4.825 Cal.

Heat produced in 6 min = 4.825 x A

Heat produced in one hour = 4.825A x 10

Units of BMR : BMR is expressed as Calories per square meter of body surface area per hour i.e. Cal/ sq.m/hr.

For the calculation of body surface area, the simple formula devised by Du Bois  is used.

A = H^0.725 x W^0.425 x 71.84

where A = Surface area in cm²

H = Height in cm

W = Weight in kg.

To convert the surface area into square meters (m²), divide the above value (cm²) by 10,000. Nomograms of body surface area (directly in m²) from heights and weights are readily available in literature.

Normal values of BMR : For adult man 35-38 Cal/sq. m/hr; for adult women 32-35 Cal/sq.m/hr.

A BMR value between -15 % and +20 % is considered as normal.

Some authors continue to represent BMR as Cal/ day. For an adult man BMR is around 1,600 Cal/day, while for an adult woman around 1,400 Cal/day. This is particularly important lor easily calculating energy requirements per day.

Factors affecting BMR

1. Surface area : The BMR is directly propor­tional to the surface area. Surface area is related to weight and height.

2. Sex : Men have marginally higher (about 5%) BMR than women. This is due to the higher proportion of lean muscle mass in men.

3. Age : In infants and growing children, with lean muscle mass, the BMR is higher. In adults, BMR decreases at the rate of about 2% per decade of life.

4. Physical activity : BMR is increased in persons (notably athletes) with regular exercise. This is mostly due to increase in body surface area.

5. Hormones : Thyroid hormones (T₃ and T₄) have a stimulatory effect on the metabolism of the body and, therefore, BMR. Thus, BMR is raised in hyperthyroidism and reduced in hypothyroidism. In fact, the measurement of BMR was earlier used to assess thyroid function.

The other hormones such as epinephrine, cortisol, growth hormone and sex hormones increase BMR.

6. Environment : In cold climates, the BMR is higher compared to warm climates.

7. Starvation : During the periods of starvation, the energy intake has an inverse relation with BMR, a decrease up to 50 % has been reported. This may be an adaptation by the body.

8. Fever : Fever causes an increase in BMR. An elevation by more than 10 % in BMR is observed for every 1 °C rise in body temperature.

9. Disease states : BMR is elevated in various infections,   leukemias,   polycythemia,   cardiac failure, hypertension etc. In Addison's disease (adrenal insufficiency) BMR is marginally lowered.

10. Racial variations : The BMR of Eskimos is much higher. The BMR of Oriental women living in USA is about 10 % less than the average BMR of American women.

Significance of BMR

BMR is important to calculate the calorie requirement of an individual and planning of diets. Determination of BMR is useful for the assessment of thyroid function. In hypothyroidism BMR is lowered (by about -40%) while in hyperthyroidism it is elevated (by about +70%). Starvation and certain disease conditions also influence BMR (described above).

SPECIFIC DYNAMIC ACTION

The phenomenon of the extra heat production by the body, over and above the calculated caloric value, when a given food is metabolized by the body, is known as specific dynamic action (SDA). It is also known as calorigenic action or thermogenic action or thermic action of food.

SDA for different foods : For a food containing 25 g of protein, the heat production from the caloric value is 100 Cal (25 x 4 Cal). However, when 25 g protein is utilized by the body, 130 Cal of heat is liberated. The extra 30 Cal is the SDA of protein. Likewise, consumption of 100 Cal of fat results in 113 Cal and 100 Cal of carbohydrate in 105 Cal, when metabolized in the body. SDA for protein, fat and carbohydrate are 30%, 13% and 5%, respectively. Thus, proteins possess the highest SDA while carbohydrates have the lowest.

SDA for mixed diet : For a mixed diet, the SDA is not an additive value of different foods but it is much less. The presence of fats and carbohydrates reduces the SDA of proteins. Fats are most efficient in reducing SDA of foodstuffs. For a regularly consumed mixed diet, the SDA is around 10%.

Significance of SDA : For the utilization of foods by the body, certain amount of energy is consumed from the body stores. This is actually an expenditure by the body for the utilization of foodstuffs. It is the highest for proteins (30%) and lowest for carbohydrates (5%) and for a mixed diet around 10%. It is, therefore, essential that an additional 10% calories should be added to the total energy needs (of the body) towards SDA. And the diet should be planned, accordingly. SDA is quite comparable to the handling charges levied by a bank for an outstation chequel.

The higher SDA for protein indicates that it is not a good source of energy. Fat is the best source of energy due to  its  lowering effect on  SDA.

However,   excessive  utilization   of  fat   leads  to ketosis.

Mechanism of SDA : The exact cause of SDA is not known. It is generally believed that SDA of foods is due to the energy required for digestion, absorption, transport, metabolism and storage of foods in the body.

Intravenous administration of amino acids or the oral ingestion of proteins gives the same SDA. This shows that the SDA of proteins is not due to their digestion and absorption. Hepatectomy abolishes SDA, thereby indicating that SDA is closely connected with the metabolic functions of liver. The SDA of proteins is primarily to meet the energy requirements for deamination, synthesis of urea, biosynthesis of proteins, synthesis of triacylglycerol (from carbon skeleton of amino acids). It has been demonstrated that certain amino acids (phenylalanine, glycine and alanine) increase the SDA. It is a common experience that consumption of a protein rich diet makes us feel warm and comfortable in cold weather. This is due to the high SDA of proteins.

The SDA of carbohydrates is attributed to the energy expenditure for the conversion of glucose to glycogen.

As regards fat, the SDA may be due to its storage, mobilization and oxidation.

PHYSICAL ACTIVITY OF THE BODY

The physical activity of the individual is highly variable. The amount of energy needed for this depends mainly on the duration and intensity of muscular activity. The expenditure of energy for the various physical activities has been calculated.

For the sake of convenience, the individuals are grouped into four categories with regard to their physical activity and the requirement of energy.

Light work            —    30 – 40 % of BMR (teachers, office workers, doctors)

Moderate work     —   40 – 50 % of BMR (housewives, students)

Heavy work            -   50 – 60 % of BMR (agricultural labourers, miners)

Very heavy work     —   60 - 100 % of BMR (construction workers, rikshaw pullers)

 

Type of physical activity and energy expenditure

(over and above BMR, about 65 Cal/kr).

Physical activity                 Energy requirement

             (Cal/hr)

Sitting (quietly)                                25

Standing (quietly)                            30

Writing/eating/reading                     30

Car driving                                        60

Typing                                              75

Household work (dish washing)     80

Walking (slow)                               130

Sexual intercourse                          140

Cycling (slow)                                150

Running (moderate)                           500

Swimming                                       600

Walking upstairs                            800

 

Energy requirements of man

As already stated, the three factors – basal metabolic rate, specific dynamic action and physical activity – determine the energy needed by the body. In an individual with light work, about 60% of the calories are spent towards BMR, about 30% for physical activity and about 10% to take care of the SDA.

The daily requirement of energy is rather variable which depends on the BMR (in turn depends on age, sex, body size etc.) and physical activity. As per some rough calculation, caloric requirements of an adult per day (Cal/day) is in the following ranges.

Light work               –   2,200-2,500

Moderate work        –   2,500-2,900

Heavy work             –   2,900-3,500

Very heavy work      –   3,500-4,000

NUTRITIONAL IMPORTANCE OF CARBOHYDRATES

Dietary carbohydrates are the chief source of energy. They contribute to 60-70% of total caloric requirement of the body. Carbohydrates are the most abundant dietary constituents, despite the fact that they are not essential nutrients to the body. From the nutritional point of view, carbohydrates are grouped into 2 categories:

1.   Carbohydrates utilized by the body—starch, glycogen, sucrose, lactose, glucose, fructose etc.

2.   Carbohydrates not utilized (not digested) by the body—cellulose, hemicellulose, pectin, gums etc.

Among the carbohydrates utilized by the body, starch is the most abundant. The consumption of starch has distinct advantages due to its bland taste, satiety value and slow digestion and absorption. Sucrose (the table sugar), due to its sweetness, can be consumed to a limited extent. Excessive intake of sucrose causes dental caries and an increase in plasma lipid levels and is associated health complications.

Functions of carbohydrates:

1.   Major sources of energy : Carbohydrates are the principal source of energy, supplying 60-80% of the caloric requirements of the body. The actual intake of carbohydrate is dependent on the food habits and economic status of the individual.

2.   Protein sparing action : Proteins perform a specialized function of body building and growth. The wasteful expenditure of proteins to meet the energy  needs  of the  body  should  be  curtailed. Carbohydrates come to the rescue and spare the proteins from being misused for caloric purpose.

3.   Absolute requirement by brain : The brain and other parts of central  nervous system  are dependent   on   glucose   for   energy.    Prolonged hypoglycemia   may   lead   to   irreversible   brain damage.

4.   Required for the oxidation of fat : Acetyl CoA is the product formed in fatty acid oxidation. For its further oxidation via citric acid cycle, acetyl CoA   combines   with   oxaloacetate,   the   latter  is predominantly derived from carbohydrates. It may therefore   be   stated   'Fat  burns   in   a   fuel  of carbohydrate'. Excess utilization of fats coupled with deficiency of carbohydrates leads to ketosis.

5.   Energy supply for muscle work : The muscle glycogen is broken down to lactic acid (glycolysis) to provide energy for muscle contraction.

6.   Synthesis of pentoses : Pentoses (e.g. ribose) are the constituents of several compounds in the body e.g. nucleic acids (DNA, RNA), coenzymes (NAD\ FAD). These pentoses  are  produced  in carbohydrate metabolism.

7.   Synthesis of non-essential amino acids : The intermediates of carbohydrate metabolism, mainly the keto acids (e.g. pyruvic acid), serve as pre­cursors for the synthesis of non-essential amino acids.

8.   Synthesis of fat : Excess consumption of carbohydrates leads to the formation of fat which is stored in the adipose tissue.

9.   Special functions in liver : Liver is the central organ   that   integrates   the   body   metabolisms. Carbohydrates play an active role in this metabolic integration The liver also utilizes certain products of carbohydrate metabolism (e.g. glucuronic acid) for detoxification.

10.Importance    of   non-digestible    carbo­hydrates : These are the carbohydrates not utilized by the body. Yet, they are important since they improve bowel motility, prevent constipation, lower cholesterol    absorption    and    improve    glucose tolerance.

Sources of carbohydrates

Carbohydrates are abundant in several naturally occurring foods. These include table sugar (99%), cereals (60-80%), pulses (50-60%), roots and tubers (20-40%) and bread (50-60%).

Requirement of carbohydrates

In a well balanced diet, at least 40 % (400-500 g /day) of the caloric needs of the body should be met from carbohydrates.

FIBER IN NUTRITION

The complex carbohydrates that are not digested by the human enzymes are collectively referred to as dietary fiber. These include cellulose, hemicellulose, pectin, lignin, gums and mucilage. It may, however, be noted that some of the fibers are digestible by the enzymes of intestinal bacteria (e.g. pectins, gums). For a long time, fiber was regarded as nutritional waste. And now nutritionists attach a lot of importance to the role of fiber in human health. Dietary fiber is involved in several functions.

Beneficial effects of fiber

1.   Prevents   constipation   :   Fiber   helps   to maintain the normal motility of gastrointestinal tract (GIT) and prevents constipation.

2.   Eliminates bacterial toxins : Fiber adsorbs large   quantities   of  water   and   also   the   toxic compounds produced by intestinal bacteria that lead   to   increased   fecal   mass   and   its   easier expulsion.

3.   Decreases GIT cancers : The lower incidence of cancers of gastrointestinal tract (e.g. colon and rectum) in vegetarians compared to non-vegetarians is attributed to dietary fiber.

4. Improves glucose tolerance : Fiber improves glucose tolerance by the body. This is mainly done by a diminished rate of glucose absorption from the intestine.

5. Reduces plasma cholesterol level : Fiber decreases the absorption of dietary cholesterol from the intestine. Further, fiber binds with the bile salts and reduces their enterohepatic circulation. This causes increased degradation of cholesterol to bile salts and its disposal from the body.

6.   Satiety value : Dietary fiber significantly adds to the weight of the foodstuff ingested and gives a sensation of stomach-fullness. Therefore, satiety is achieved   without   the   consumption   of   excess calories.

Adverse affects of fiber

Some of the food fads went to the extent of ingesting huge quantities of rice bran to achieve all the benefits of fiber. This led to several complications. In general, the harmful effects are mostly observed in people consuming large quantities of dietary fiber.

1.           Digestion   and   absorption   of   protein   is adversely affected.

2.     The intestinal absorption of certain minerals (e.g. Ca, P, Mg) is decreased.

3.           Intestinal bacteria ferment some fibers, causing flatulence and often discomfort.

Sources of dietary fiber:

Fruits, leafy vegetables, whole wheat legumes, rice bran.

NUTRITIONAL IMPORTANCE OF LIPIDS

Triacylglycerols (fats and oils) are the  concentrated dietary source of fuel, contributing 15-50% of the body energy requirements. Phospholipids and cholesterol (from animal sources) are also important in nutrition.

Major nutritional functions of lipids

Dietary lipids have two major nutritive functions.

1.   Supply     triacylglycerols     that     normally constitute about 90% of dietary lipids which is a concentrated source of fuel to the body.

2. Provide essential fatty acids and fat soluble vitamins (A, D, E and K).

ESSENTIAL FATTY ACIDS

The unsaturated fatty acids which the body cannot synthesize and, therefore, must be consumed in the diet are referred to as essential fatty acids (EFA).

The fatty acids—linoleic and linolenic acid— cannot be synthesized by humans. In a strict sense, only these two are essential fatty acids. Arachidonic acid can be synthesized from linoleic acid in some animal species, including man. However, the conversion efficiency of linoleic acid to arachidonic acid is not clearly known in man. And for this reason, some nutritionists recommend that it is better to include some amount of arachidonic acid also in the diet.

Functions of EFA

1.     Essential fatty acids are the structural components of biological membranes.

2. Participate in the transport and utilization of cholesterol.

3.   Prevent fat accumulation in the liver.

4.   Required for the synthesis of prostaglandins.

5.   Maintain proper growth and reproduction of the organisms.

Deficiency of EFA

Essential fatty acid deficiency is associated with several complications. These include impairment in growth and reproduction, increased BMR and high turnover of phospholipids. The EFA deficiency in humans is characterized by a scaly dermatitis on the posterior and lateral parts of limbs and buttocks. This condition is referred to as phrynoderma or toad skin. Poor wound healing and hair loss is also observed in EFA deficiency.

EFA content of foods

The essential fatty acids, more frequently called polyunsaturated fatty acids (PUFA), are predominantly present in vegetable oils and fish oils. The rich vegetable sources include sunflower oil, cotton seed oil, corn oil, soyabean oil etc.

The fat of animal origin (exception—fish), contain less PUFA e.g. butter, fat of meat, pork and chicken.

Dietary intake of EFA

Nutritionists recommend that at least 30 % of the dietary fat should contain PUFA. Very high intake of PUFA (i.e. totally replacing saturated fatty acids) may not be advisable. This is due to the fact that excess PUFA, unless accompanied by antioxidants (vitamin E, carotenes), is believed to be injurious to the cells due to the overproduction of free radicals.

CHOLESTEROL IN NUTRITION

It is proved beyond doubt that the elevated serum cholesterol (>250 mg/dl) increases the risk of atherosclerosis and coronary heart diseases. But the role of dietary cholesterol in this regard is still r controversial. Cholesterol synthesis continuously occurs in the body which is under a feedback regulation. Some nutritionists believe that dietary cholesterol may not have much influence on the body levels while others recommend to avoid the consumption of cholesterol rich foods (e.g. egg yolk) for a better health.

It is an accepted fact that reduction in serum cholesterol level lowers the risk of heart diseases.

REQUIREMENT OF DIETARY FAT

Consumption of dietary fats and oils is considered in terms of their contribution towards the energy needs of the body. There is a wide variation in fat intake. It is much higher (up to 50 % of daily calories) in affluent societies compared to the poorer sections of the people (about 15 % of calories). The recommended fat intake is around 20-30 % of the daily calorie requirement, containing about 50 % of PUFA.

NUTRITIONAL IMPORTANCE OF PROTEINS

Proteins have been traditionally regarded as 'body-building foods'. However, 10-15 % of the total body energy is derived from proteins. As far as possible, carbohydrates spare proteins and make the latter available for body-building process. The functions carried out by proteins in a living cell are innumerable, a few of them are listed here.

Functions of proteins

1. Proteins are the fundamental basis of cell structure and its function.

2. All the enzymes, several hormones, immunoglobulins, transport carriers etc., are proteins.

3. Proteins are involved in the maintenance of osmotic   pressure,   clotting   of   blood,    muscle contraction etc.

4. During  starvation,   proteins  (amino  acids) serve as the major suppliers of energy. It may be noted that the structural proteins themselves serve as 'storage proteins' to meet the emergency energy needs of the body. This is in contrast to lipids and carbohydrates which have the respective storage forms   triacylglycerols   (in   adipose   tissue)   and glycogen (in liver and muscle)

Essential amino acids

The nutritional importance of proteins is based on the content of essential amino acids. There are ten essential amino acids – arginine, valine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and threonine (code to recall—AV HILL MP TT). Of these two—namely arginine and histidine—are semi-essential. The requirement of 8 essential amino acids per kg body weight per day is given in Table. Cysteine and tyrosine can, respectively, spare the requirement of methionine and phenylalanine.

Requirements of essential amino acids

Amino acid          Requirement

                                  (mg/kg body weight/day)

Valine                       14

Isoleucine                 12

Leucine                      16

Lysine                       12

Methionine*               10

Phenylalanine*        16

Tryptophan                 3

Threonine                  8

* Cysteine and tyrosine can, respectively, spare (partly) the require­ment of methionine and phenylalanine.

NITROGEN BALANCE

Dietary protein is almost an exclusive source of nitrogen to the body. Therefore, the term nitro­gen balance truly represents the protein (16 % of which is nitrogen) utilization and its loss from the body.

Nitrogen balance is determined by comparing the intake of nitrogen (chiefly by proteins) and the excretion of nitrogen (mostly undigested protein in feces; urea and ammonia in urine). A normal healthy adult is in a nitrogen equilibrium since the daily dietary intake (I) is equal to the loss through urine (U), feces (F) and sweat (S).

I = U + F + S

Thus, an individual is said to be in a nitrogen balance if the intake and output of nitrogen are the same (Fig. 23.1). There are two other situations – a positive and a negative nitrogen balance. Positive nitrogen balance : This is a state in which the nitrogen intake is higher than the output. Some amount of nitrogen is retained in the body causing a net increase in the body protein. Positive nitrogen balance is observed in growing children, pregnant women or during recovery after serious illness.

Negative nitrogen balance : This is a situation in   which the nitrogen output is higher than the input. The result is that some amount of nitrogen is lost from   the   body   depleting   the   body   ptotein.  Prolonged  negative  nitrogen balance  may  even lead  to  death.  This   is  sometimes  observed   in children suffering from kwashiorkor or marasmus. Negative nitrogen balance may occur due to inadequate dietary intake of protein (deficiency of even a single essential amino acid) or destruction of tissues or serious illness. In all these cases, the body adapts itself and increases the breakdown of

tissue proteins causing loss of nitrogen from the body.

 

Other factors influencing nitrogen balance

Besides the major factors discussed above (growth, pregnancy, protein deficiency, injury illness) several other factors influence nitroger balance.

Hormones : Growth hormone and insulin pro­mote positive nitrogen balance while corticosteroids result in negative nitrogen balance.

Disease states : Cancer and uncontrollec diabetes cause negative nitrogen balance.

ASSESSMENT OF NUTRITIVE VALUE OF PROTEINS

Knowledge on the quantity of dietary protein alone is not sufficient to evaluate the nutritional importance of proteins. This is in contrast to dietary carbohydrates and lipids. The quality of the proteins which depends on the composition of essential amino acids is more important. Several laboratory methods are in use to assess the nutritive value of proteins. Of these, four methods—protein efficiency ratio, biological value, net protein utilization and chemical scove—are discussed briefly.

Protein efficiency ratio (PER)

This test consists of feeding weaning (21 day old) albino rats with a 10 % test protein diet and recording the gain in body weight for a period of 4 weeks. PER is represented by gain in the weight of rats per gram protein ingested.

PER =       Gain in body weight (g) / Protein ingested   (g)

The PER for egg protein is 4.5; for milk protein 3.0; for rice protein 2.2.

Biological value

The biological value (BV) of protein is defined as the percentage of absorbed nitrogen retained in the body.

BV =     Nitrogen retained / Nitrogen absorbed × 100

For the measurement of BV, the experimental animals, namely weaning albino rats are chosen. They are first fed with a protein-free diet for 10 days. Then they are kept on a 10 % protein diet to be tested for BV. Urine and feces are collected for both the periods i.e. protein-free diet and protein diet. Nitrogen is estimated in the diet, feces and urine samples. Biological value can be calculated by the following formula

    (N absorbed - N lost in metabolism)

BV = ------------------------- -------------------- x100

                             N absorbed

 

    [I_n – (F_n  – F_c)] – (U_n  – U_c)

BV = ------------------------- -------------------- x100

                             I_n – (F_n  – F_c)

where   I_n   =   Nitrogen ingested

    F_n  =      Nitrogen in   feces (on protein diet)

    F_c  =    Nitrogen in feces (on protein-free diet)

    U_n =    Nitrogen in urine (on protein diet)

    U_c =    Nitrogen in urine (on protein-free diet)

For the calculation of BV of proteins, experiments can be done even in human subjects. The BV for different protein sources is given in Table 23.4.

The biological value provides a reasonably good index for the nutritive value of proteins. But unfortunately this method has an inherent drawback. It cannot take into account the nitrogen that might be lost during the digestion process. For instance, if the ingested nitrogen is 100 mg, absorbed is 10 mg and retained is 8 mg, the BV 8/10 x100 = 80. This figure is erroneous, since the major part of the protein (90 mg) did not enter the body at all for utilization.

Net protein utilization (NPU)

This is a better nutritional index than biological value, since it takes into account the digestibility factor. The experimental procedure for NPU is similar to that of BV. Net protein utilization can be calculated as

NPU = Nitrogen retained / Nitrogen ingested x100

Chemical score

This is based on the chemical analysis of the protein for the composition of essential amino acids which is then compared with a reference protein usually egg protein). The chemical score is defined as the ratio between the quantitity of the most limiting essential amino acid in the test protein to he quantity of the same amino acid in the egg protein, expressed as percentage.

Chemical score =  mg of the limiting amino acid /g test protein      x 100

                                    mg of the same amino acid /g egg protein

 

The chemical score of egg protein, for any one of the essential amino acids, is taken as 100 and the rest of the proteins are compared.

Mutual supplementation of proteins

As is observed from the Table 23.4, the animal proteins are superior in their nutritive value compared to the proteins of vegetable origin. Further, some of the essential amino acids are limiting in certain foods. For instance, rice and wheat proteins are limiting in lysine and threonine while the protein of Bengal gram is limited in sulfur-containing amino acids (methionine and cystine).

It is fortunate that humans (worldover) have the habit of consuming a mixed diet, with different foods, simultaneously. This helps to overcome the deficiency of certain essential amino acids in one food by being supplemented from the others. This phenomenon   is  referred   to  as  mutual supple­mentation. For instance, an Indian diet with cereals (wheat, rice) is taken along with pulses (dal). The limitation of lysine and threonine in cereal proteins is overcome by their supplementation  from  dal proteins. Simultaneously, the limitation of sulfur containing amino acids in dal is also compensatec by the cereals which are rich in them.

The nutritive value of protein of a particula food can be enhanced by appropriate combination with other foods. Due to the consumption of mixed diets, dietary deficiency of essential amino acids is most uncommon. Further, the principle of mixed diet takes care to supply adequate quantities of essential amino acids to the people subsisting oi pure vegetarian diets. It has to be remembered thet the effect of mutual supplementation in proteins is best observed with the same meal (or at least on the same day).

Requirement of proteins

The requirement of protein is dependent on its nutritive value,  caloric  intake and  physiological  states (growth, pregnancy, lactation) of the individual. For an adult, 0.8-1.0 g protein/kg body weight/day is adequate. The requirement should be nearly double for growing children, pregnant and lactating women.

Dietary sources of proteins

The protein content of foods is variable, cereals have 6-12 %; pulses 18-22 %; meat 18-25 %, egg 10-14 %; milk 3-4 % and leafy vegetables 1-2 %. In general, the animal proteins are superior than vegetable proteins as the dietary source.

NUTRITIONAL IMPORTANCE OF VITAMINS AND MINERALS

Vitamins are a group of organic nutrients required in small quantities for a variety of biochemical functions and which, generally, cannot be synthesized by the body and must therefore be supplied in the diet. The lipid-soluble vitamins are apolar hydrophobic compounds that can only be absorbed efficiently when there is normal fat absorption. They are transported in the blood, like any other apolar lipid, in lipoproteins or attached to specific binding proteins. They have diverse functions, eg, vitamin A, vision; vitamin D, calcium and phosphate metabolism; vitamin E, antioxidant; vitamin K, blood clotting. As well as dietary inadequacy, conditions affecting the digestion and absorption of the lipid-soluble vitamins—such as steatorrhea and disorders of the biliary system—can all lead to deficiency syndromes, including: night blindness and xerophthalmia (vitamin A); rickets in young children and osteomalacia in adults (vitamin D); neurologic disorders and anemia of the newborn (vitamin E); and hemorrhage of the newborn (vitamin K). Toxicity can result from excessive intake of vitamins A and D. Vitamin A and β-carotene (provitamin A), as well as vitamin E, are antioxidants and have possible roles in atherosclerosis and cancer prevention.

The water-soluble vitamins comprise the B complex and vitamin C and function as enzyme cofactors. Folic acid acts as a carrier of one-carbon units. Deficiency of a single vitamin of the B complex is rare, since poor diets are most often associated with multiple deficiency states. Nevertheless, specific syndromes are characteristic of deficiencies of individual vitamins, eg, beriberi (thiamin); cheilosis, glossitis, seborrhea (riboflavin); pellagra (niacin); peripheral neuritis (pyridoxine); megaloblastic anemia, methylmalonic aciduria, and pernicious anemia (vitamin B₁₂); and megaloblastic anemia (folic acid). Vitamin C deficiency leads to scurvy.

 

 

 

Inorganic mineral elements that have a function in the body must be provided in the diet. When the intake is insufficient, deficiency symptoms may arise, eg, anemia (iron), cretinism and goiter (iodine). If present in excess as with selenium, toxicity symptoms may occur.

 

      1. Much smaller amounts of inorganic trace elements are required if these are being provided as part of TPN.

2.       The total concentration in iodine-containing compounds in plasma, mainly contained in the thyroid hormones,  = 250-600 nmol/L; only 5 nmol/L is present as inorganic iodide.

3.       10-20 mg/day in the reproductive period; 20-50 mg/day during pregnancy.

 

RECOMMENDED DIETARY ALLOWANCES (RDA)

The recommended dietary allowances (RDA) represents the quantities of the nutrients to be provided in the diet daily for maintaining good health and physical efficiency of the body. It must be remembered that RDA is not the minimum amount to just meet the body needs, but allowance is given for a safe margin.

Factors affecting RDA

Sex   :   The   RDA   for   men   is  about   20 % higher than that for women. Iron is an exception as   the   requirement   is   greater   in   menstruating  women. Additional  requirements (20-30 %  above normal) are needed  for pregnant and  lactating women.

Age : In general, the nutrient requirement is much higher in the growing age. For instance, the protein requirement for a growing child is about 2 g/kg body wt/day compared to 1 g/kg body wt/ day for adults.

RDA for adult man

For a quick recapitulation, the RDA of macronutrients (carbohy­drate, fat and protein) and selected micronutrients (vitamins and minerals) for an adult man weighing 70 kg are given in Table 23.5.

BALANCED DIET

After discussing the nutritional aspects of dietary ingradients and their RDA, it is worthwhile to formulate a diet for man. A balanced diet is defined as the diet which contains different types of foods, possessing    the    nutrients – carbohydrates,    fats,  proteins, vitamins and minerals – in a proportion to meet the requirements of the body. A balanced diet invariably supplies a little more of each nutrient than the minimum requirement to withstand the short duration of leanness and keep the body in a state of good health.

The basic composition of balanced diet is highly variable, as it differs from country to country, depending on the availability of foods. Social and cultural habits, besides the economic status, age, sex and physical activity of the individual largely influence the intake of diet.

The Nutrition Expert Croup, constituted by the Indian Council of Medical Research has recommended the composition of balanced diets for Indians. This is done taking into account the commonly available foods in India. The composition of balanced diet (vegetarian and non-vegetarian), for an adult man and an adult woman are, respectively, shown in tables.

The Indian balanced diet is composed of cereals (rice, wheat, jowar), pulses, vegetables, roots and tubers, fruits, milk and milk products, fats and oils, sugar and groundnuts. Meat, fish and eggs are present in the non-vegetarian diets. In case of vegetarians, an additional intake of milk and pulses is recommended.

Balanced diet for an adult man

 

 

Balanced diet for an adult woman

 

NUTRITIONAL DISORDERS

While the people of developing countries suffer from undernutrition, ovenutrition is the major concern of the developed countries. Some of the important nutritional diseases are discussed hereunder.

Protein-energy malnutrition

Protein-energy malnutrition (PEM) – sometimes called protein-calorie malnutrition (PCM)—is the most common nutritional disorder of the developing countries. PEM is widely prevalent in the infants and pre-school children. Kwashiorkor and marasumus are the two extreme forms of protein-energy malnutrition.

 

Kwashiorkor

The term kwashiorkor was introduced by Cicely Williams (1933) to a nutritional disease affecting the people of Gold Coast (modern Ghane) in Africa. Kwashiorkor literally means sickness of the deposed child i.e. a disease the child gets when the next baby is born.

Occurrence and causes : Kwashiorkor is predominantly found in children between 1-5 years of age. This is primarily due to insufficient intake of proteins, as the diet of a weaning child mainly consists of carbohydrates.

Clinical symptoms : The major clinical manifestations of kwashiorkor include stunted growth, edema (particularly on legs and hands), diarrhea, discoloration of hair and skin, anemia, apathy and moonface.

Biochemical manifestations : Kwashiorkor is associated with a decreased plasma albumin concentration (<2 g/dl against normal 3-4.5 g/dl), fatty liver, deficiency of K⁺ due to diarrhea. Edema occurs due to lack of adequate plasma proteins to maintain water distribution between blood and tissues. Disturbance in the metabolism of carbo­hydrate, protein and fat is also observed. Several vitamin deficiencies occur. Plasma retinol binding protein (RBP) is reduced. The immunologicaf responses of the child to infection is very low.

Treatment : Ingestion of protein-rich foods or the dietary combinations to provide about 3-4 g of protein/kg body weight/day will control kwashiorkor. The treatment can be monitored by measuring plasma albumin concentration, disappearance of edema and gain in body weight.

 

Marasumus

Marasmus literally means 'to waste'. It mainly occurs in children under one year age. Marasmus is predominantly due to the deficiency of calories. This is usually observed in children given watery gruels (of cereals) to supplement the mother's breast milk.

The symptoms of marasmus include growth retardation, muscle wasting (emaciation), anemia and weakness. A marasmic child does not show edema or decreased concentration of plasma albumin. This is the major difference to distinguish marasmus from kwashiorkor.

 

 

 

FUNCTIONAL AND SCREENING TESTS IN GASTROENTEROLOGY

 

 

 

 

 

Biochemical tests play a relatively minor role in the investigation of GI tract disease, and a number of  previously well-established investigations have now become obsolete. In general, microbiological investigations, radiological investigations, endoscopy and biopsy procedures have more to offer. The tests that have proved most valuable and reliable for the investigation of some

conditions are given in Table 8.1.

 

 

 

Stomach

Peptic ulcer

Most disorders of gastric function are best assessed initially   using   radiological   investigations   and endoscopy. Most peptic ulcers are associated with Helicobacter pylori

 infection which weakens the protective mucous coating of the stomach and duodenum. The organism is present in the mucosa and is protected from stomach acidity by the creation of a more neutral microenvironment through the secretion of large amounts of urease and the subsequent conversion of urea to ammonia and carbon dioxide. This reaction forms the basis of the urea breath test to detect H. pylori infection. In the few patients who present with atypical or recurrent peptic ulceration that is resistant to treatment with H₂ antagonists, proton pump inhibitors and antibiotics to eradicate H. pylori, biochemical tests to quantify plasma [gas­trin] may be of value.

 

Tests for H. pylori infection

 

Urea breath test This non-invasive test relies on the urease activity of H. pylori to detect active infection. The patient ingests either ¹³C- or ¹⁴C-labelled urea and urease, if present, hydrolyses urea into ammo­nia and isotopically labelled carbon dioxide. Carbon dioxide is absorbed from the gut and subsequently expired in the breath where it can be trapped and quantified. This breath test is used both for the iden­tification of patients with active infection and for establishing the effectiveness of treatment.

Serological tests Patients infected with H. pylori develop antibodies to the organism that can be detected in the laboratory using enzyme-linked immunosorbent assays (ELISAs). While serological tests are used to identify patients who have been infected with the organism, they are less helpful in confirming its eradication because of the slow reduction in antibody titres.

Faecal antigen testing Enzyme immunoassays can be used to detect the presence of H. pylori in stool specimens.

 

Gastrin

Gastrin is a polypeptide released by the G cells in the gastric antrum and duodenum and is a potent stimulator of gastric acid production. Its release is normally inhibited if the gastric pH is low, but cir­culating levels are increased in patients with chronic hypochlorhydria. Thus, plasma [gastrin] may be elevated as a physiological response to achlorhydria or hypochlorhydria due to gastritis, treatment with H₂ antagonists, proton pump inhibitors, pernicious anaemia or previous vago-tomy. Increased plasma [gastrin] may also be found in patients with hypercalcaemia or following gastric surgery, as a result of which the antral mucosa may have become isolated from gastric contents. The most important clinical application for the measurement of gastrin is in the investigation of patients with gastric acid hypersecretion thought to be caused by a gastrinoma (Zollinger-Ellison syndrome).

Zollinger-Ellison syndrome

This syndrome is due to a gastrinoma, that is neoplasia of either pancreatic gastrin-producing cells or gastric gastrin-producing cells, the former being the more common site. Approximately 60 % of gastrinomas are malignant and 30 % occur as part of the MEN syndrome. Increased gastrin production leads to chronic hyper secretion of gastric acid, which in turn causes peptic  ulceration and sometimes diarrhoea and fat malabsorption leading to steatorrhoea. The steatorrhoea is thought to be due to high [H⁺] in the intestine lumen; this inhibits the action of pancreatic lipase.  In some patients' an isolated simple duodenal ulcer or diarrhoea may be the presenting feature.

The diagnosis of gastrinoma is based on the detec­tion of an unequivocally elevated fasting plasma [gastrin] in the presence of gastric acid hypersecretion. Patients should not be receiving proton pump inhibitors or H₂ receptor blockers at the time of measurement. Provocative testing may be necessary in about 15 % of patients where the basal plasma [gastrin] concentration is normal or only slightly increased and gastrinoma is suspected. The pre­ferred test involves the IV injection of secretin which usually produces a 2-fold increase in plasma [gastrin] in patients with gastrinoma, while no change occurs in patients with G-cell hyperplasia.

The pancreas

The pancreas is a complex gland with important endocrine and exocrine functions. Its principal endocrine role relates to the regulation of glucose metabolism through the secretion of insulin and glucagon from the islets of Langerhans and is discussed elsewhere in this volume. Pancreatic juice is produced by the exocrine tissue and released into the duodenum where it is mixed with partially digested food. It is an alkaline fluid  that contains a mixture of enzymes essential for protein, carbohydrate and lipid digestion. Secretion is induced in response to nervous stimuli, but mainly by the hormones secretin and cholecystokinin-pancreozymin (CCK-PZ). These are secreted by the all intestine in response to the entry of food.

Acute pancreatitis

Acute pancreatitis is commonly associated with gallstones or alcoholism; vascular and infective causes have also been recognised. Confirmation of the clinical diagnosis mainly depends on plasma amylase activity measurements. Plasma [calcium] may fall considerably in severe cases of acute pancreatitis, but sometimes not for a few days; it probably falls as a result of the formation of insoluble calcium salts of fatty acids in areas of fat necrosis.
Plasma amylase

Amylase   in   plasma   arises   mainly   from   the pancreas (P-isoamylase) and the salivary glands (S-isoamylase). Plasma P-isoamylase activity is a more sensitive and more specific test than total amylase for the detection of acute pancreatitis, but total plasma amylase activity is most often measured  and  is usually,  but not  always,  greatly increased in acute pancreatitis. Plasma amylase activities greater than ten times the normal value are   virtually   diagnostic   of  acute   pancreatitis.  Maximum values of more than five times the upper reference limit are found in about 50 % of cases, but are not pathognomonic of acute pancreatitis, since similarly high values sometimes occur in the afferent loop syndrome, mesenteric infarction and acute biliary tract disease, as well as in acute parotitis. Smaller and more transient increases may occur in almost any acute abdominal condition   (e.g. perforated peptic ulcer), or after injection of morphine and other drugs that cause spasm of the sphincter of Oddi. Moderate increases have also been reported in patients with DKA. In patients with acute pancreatitis, plasma amylase activity usully returns to normal within 3-5 days.

Macro-amylasaemia In this rare disorder, part of the plasma amylase activity circulates as a high molec­ular weight form which, unlike normal amylase, is not cleared by the kidney. The diagnosis may be made when the increased plasma amylase activity is found to be persistent and accompanied by a normal urinary amylase activity.

Chronic pancreatitis

Impaired secretion of pancreatic enzymes may not be evident until the disease is advanced, but may then give rise to malabsorption, especially steatorrhoea. Various methods involving either direct (inva­sive) measurements on pancreatic fluid following duodenal intubation or indirect (non-invasive) mea­sures, without the need for intubation, have been described. The biochemical function tests have proved to be of limited value, and radiology and endoscopy are usually the preferred investigations.

 

 

Direct (invasive) tests

The secretin/CCK-PZ test involves the measure­ment of bicarbonate and amylase or trypsin in duodenal fluid following stimulation of the pan­creas with IV secretin and CCK-PZ. The Lundh test assesses the pancreatic secretory response to a standard test meal of glucose, corn oil and casilan but is also dependent on extrapancreatic factors such as gastric and vagal function and endogenous secretin and cholecystokinin secretion. Abnormal results are obtained in most cases of chronic pan­creatitis, enzymatic activity and [HCO₃^-] tending to fall before there is any obvious reduction in the volume of juice.

Although these tests have been regarded as the gold standard for assessing pancreatic function with sensitivities of 90 % for the detection of chronic pancreatitis, they are time consuming, expensive and uncomfortable for the patient and are no longer routinely used.

Indirect (non-invasive) tests

Faecal elastase Elastase is a pancreas-specific enzyme that is not degraded during intestinal transport reaching concentrations in faeces that are 5-6 times higher than those of duodenal fluid. Low levels of faecal elastase are associated with pancreatic insufficiency.

Fluorescein dilaurate (pancreolauryl) test This oral tubeless test relies on the principle that pancreatic enzymes release fluorescein from fluorescein dilau­rate. Fluorescein released following oral administra­tion of fluorescein dilaurate is absorbed, partially metabolised, and excreted in the urine where it is measured. The amount of fluorescein excreted is compared with that excreted following oral admin­istration of an equivalent oral dose of fluorescein alone on the following day. Results are expressed as the ratio of fluorescein excreted after fluorescein dilaurate and after free fluorescein. A ratio of less than 20 % is considered abnormal.

A similar test that depends on the ability of pancreatic enzymes to release p-aminobenzoic acid (PABA) following the oral administration of N-benzoyl-L-tyrosyl p-aminobenzaldehyde (BT-PABA) is no longer widely used.

The sensitivities of these indirect non-invasive tests for the detection of pancreatic insufficiency are similar. They depend on a significant loss of exocrine function and are only reliable in moder­ate to severe disease. Faecal elastase offers the advantages of acceptable reliability and simplicity since only a single stool sample is required, avoid­ing the need for prolonged urine collections. It is recommended as the test of choice in the investi­gation of patients presenting with diarrhoea thought to be of pancreatic origin.

Small intestine

In small intestinal disease, absorptive function may be diminished, and permeability (via inter­cellular junctions) is often increased. Biochemical tests are available that assess absorptive function and intestinal permeability, but the availability of jejunal biopsy has greatly reduced the need to perform such tests for diagnostic purposes. These tests may also be used to monitor the response to therapy (e.g. the response of patients with coeliac disease to a gluten-free diet).

Tests of carbohydrate absorption

Xylose absorption test

D-Xylose, a pentose, is normally absorbed rapidly from the small intestine and excreted in the urine little is metabolised in the liver. Its concentration in blood or excretion in urine following a standard oral dose of xylose has been used to investigate the intestine's   ability  to   absorb   monosaccharides.  Impaired   absorption   and   excretion   of  xylose occurs in patients with disease of the small intes­tine but low values may also be observed ii patients who have bacterial colonisation of the small intestine, since the bacteria may metabolise xylose. Also, low urinary values occur in patients with renal disease, due to impaired excretion of xylose. Although some laboratories continue to offer the xylose absorption test, it has been large, superceded by the widespread availability of sma bowel histology following endoscopy.

Disaccharidase deficiency

Disaccharidase deficiency may be exhibited as intol­erance to one or more of the disaccharides – lactose, maltose or sucrose. The defect may be congenital or acquired. Disaccharidase activity can be measured in small intestinal mucosa biopsy specimens. This is the most reliable way of specifically diagnosising small intestinal disaccharidase deficiency.

Tests of intestinal permeability

Intestinal permeability can be assessed by giving [⁵¹Cr]-EDTA (100 mCi) by mouth and then collect­ing urine for 24 h. Increased urinary excretion of [⁵¹Cr]-EDTA occurs in intestinal disease. Glomerular function should be assessed before performing this test, as its interpretation depends on there being normal glomerular function. An alternative approach involves a sugar permeability test in which a mixture of disaccharide (e.g. lactulose) and monosaccharide (e.g. rhamnose) is administered orally and the differential urinary excretion gives an index of intestinal permeability. While these tests provide very sensitive indices of intestinal perme­ability, they are non-specific and non-diagnostic other than demonstrating a mucosal abnormality. Although these tests have been widely used in the research setting, they are rarely performed in routine finical practice.

Amino acid absorption

Certain specific disorders of amino acid transport affect both intestinal and renal epithelial trans­port. In Hartnup disease, there is impaired transport of neutral amino acids, and deficiency of some essential amino acids (especially tryptophan) may occur. In cystinuria, the dibasic amino acids cystine, ornithine, arginine and lysine) are affected; however, there is no associated nutritional defect, despite the fact that lysine is an essential amino acid. These disorders are investigated by examining the pattern of amino acids excreted in the urine by chromatography.

Fat absorption

Efficient digestion and absorption of fat requires both effective emulsification and solubilisation of

fats and lipolysis, followed by the absorption of hydrolysed products across the jejunum. Bile acids play an important role in this process.

The primary bile acids cholic acid and chenodeoxycholic acid are formed in the liver from cholesterol, conjugated with glycine and taurine, and then excreted in bile. Most are reabsorbed unchanged by an active process in the terminal ileum, and are transported back to the liver where they are re-excreted in bile, thus complet­ing the enterohepatic circulation (Figure 8.1).

 

Approximately one-quarter of primary bile acid conjugates are deconjugated by intestinal bacteria but are subsequently reabsorbed and completely reconjugated in the liver. The secondary bile acid, deoxycholic acid, formed by bacterial action on cholic acid in the gut is also absorbed in the terminal ileum and conjugated with glycine or taurine in the liver prior to being excreted in bile.

Bile salts are amphipathic molecules (i.e. they contain both hydrophobic and hydrophilic groups) and have a detergent-like action that causes the emulsification of fat globules. They also act as lipid carriers and solubilise lipids through the formation of micelles allowing the products of lipolysis to be absorbed. Bile salts also promote the action of pancreatic lipase and co-lipase.

During the absorption of a fat-containing meal, conjugated bile acids must be present in the upper small intestine in concentrations sufficient to allow the formation of micelles. Insufficient con­centrations of conjugated bile acids may give rise to malabsorption of fat (Table 8.2).

 

Fat-soluble vitamins (A, D, E and K) share absorptive mechanisms with other dietary lipids. Malabsorption of fat-soluble vitamins, which is most commonly manifested as vitamin D defi­ciency, occurs in conditions causing fat malabsorption.

Determination of fat absorption

Faecal fat

Three- or five-day collections of stools for the mea­surement of unabsorbed fat and the assessment of malabsorption have been used for decades and continue to be requested. However, due to difficul­ties relating to the inherently unpleasant nature of the test, inadequate sample collection, lack of ana­lytical quality control and standardisation, and the limited diagnostic information provided by a posi­tive result, it has been suggested that the routine use of this test should be abandoned. If performed, the patient should consume 90-100 g of fat per day during the collection. Fat excretion in a nor­mal individual is <18 mmol/24 h.

Triglyceride (triolein) breath test This test avoids the difficulties and unpleasantness of collecting faeces over several days. Following digestion and absorption of an oral dose of [¹⁴C]-triolein (the marker is in the fatty acid component), part of the fatty acid is metabolised to ¹⁴CO₂, which is then excreted in expired air. A high ¹⁴CO₂ excre­tion is associated with normal fat absorption whereas ¹⁴CO₂ excretion is low in patients with fat malabsorption. Similar fat absorption tests based on stable isotopes have also been developed using a variety of substrates. Breath tests have a low sensi­tivity for mild or moderate malabsorption.

Bacterial colonisation of the small intestine

The small intestine is usually sterile. However when there is stasis (e.g. blind loop, stricture) or a colonic fistula or, occasionally, when immune mechanisms are impaired, anaerobic bacteria colonise the intestine. This often causes fat malab­sorption, due at least partly to deconjugation of bile acid conjugates by the bacteria. Vitamin B₁₂ deficiency may also develop due to its consump­tion by the bacteria.

Diagnosis of bacterial colonisation of the small intestine requires intubation for the collection of specimens, on which microbiological procedures are then performed. However, some non-invasive tests have been devised for detecting the possible presence of bacterial colonisation.

Breath tests

The glucose hydrogen breath test is based on the ability of some bacteria to ferment carbohydrates with an end product of hydrogen, which is not Traduced by mammalian cells. The hydrogen pro­duced in the gut by bacterial action following an :ral glucose load is absorbed from the intestine and transported to the lungs where it is excreted .n expired air and can be measured. Similarly, the ¹⁴C]-xylose breath test depends on the ability of inaerobic bacteria to metabolise [¹⁴C]-xylose with the production of ¹⁴CO₂, which can also be quantified in expired air. The sensitivities of both tests ire poor compared with the culture of a small bowel aspirate but are of value if a positive result is obtained. The simpler glucose hydrogen breath test is recommended.

Terminal ileal function

Bile salts and vitamin B₁₂ are absorbed in the terminal ileum, and tests are available that assess the absorption of these compounds. The Schilling test is used to assess vitamin B₁₂ absorption, and is usually performed in haematology laboratories. The test is abnormal in patients with pernicious anaemia and in patients with disease of the terminal ileum.

Evidence of bile acid malabsorption can be obtained  by  the   measurement  of  the   serum metabolite    7a-hydroxy-4-cholesten-3-one,     an intermediate in the bile acid biosynthetic pathway, which is increased in the presence of increased bile acid turnover. While this test is not widely available at present, it has the potential to replace the more expensive 75Se-homotaurocholate (75Se-HCAT) test in which the percentage retention of  an oral dose of this synthetic gamma-emitting bile salt is estimated by whole body scanning, 7 days after its administration.

Reabsorption of water and inorganic constituents

About 8 L of intestinal secretions are produced each day, and if these are not largely reabsorbed, fluid and electrolyte disturbances will develop rapidly. Reabsorption takes place not only in the jejunum and ileum, but also in the colon. Acute and severe disturbances may occur in patients following surgery, especially operations on the GI tract, and losses of K⁺ often become very large.

Non-surgical intestinal causes of electrolyte imbalance include severe diarrhoea (e.g. due to cholera, in which there is a defect in Na⁺ reabsorption in the jejunum).

Serological tests for coeliac disease

Coeliac disease is the most common small bowel enteropathy in the Western world with a prevalence of between 1 : 200 and 1 : 159 in European and North American populations. Untreated patients with coeliac disease have IgA anti-endomysium and anti-tissue transglutaminase antibodies. These sero­logical markers have a high sensitivity and speci­ficity for coeliac disease and are useful for both screening and monitoring the response to treat­ment. They are also the most frequently requested laboratory tests for the investigation of patients with GI disturbances. However, it should be noted that selective IgA deficiency occurs in approxi­mately 2-3 % of patients with coeliac disease and, to avoid false-negative results, total IgA levels should be measured in patients undergoing initial screen­ing. Although these tests are very efficient at detect­ing the antibodies involved in the immune response to coeliac disease, a small intestinal biopsy is mandatory to make the final diagnosis.

Gastrointestinal inflammation

Calprotectin is a calcium-binding protein derived from activated neutrophils as a result of inflamma­tion and it is elevated in faeces when pathology resulting in an inflammatory process occurs in the intestine. Highest levels are found in inflammatory bowel disease and bacterial infection, but faecal cal­protectin may also be increased in cancer of the colon and stomach, colonic polyps and diverticular disease. In contrast, faecal calprotectin is normal in patients with irritable bowel syndrome in which there is no identifiable pathology in the intestine. Available evidence indicates that faecal calpro­tectin has a high sensitivity and specificity for

organic disease and that a negative result has the potential to reduce the need for further expensive GI investigations. Currently, the calprotectin assay is available in specialised centres only, but its use will undoubtedly become more widespread if the initial potential of the test is fulfilled.

The investigation of malabsorption and diarrhoea

Efficient digestion and absorption require the stom­ach, pancreas, hepatobiliary system and small intes­tine all to be functioning normally. Severe defects in the function of any one of these organs may cause intestinal malabsorptive disease; the patient may complain of diarrhoea or weight loss. The causes of carbohydrate, protein and amino acid, and lipid malabsorption are summarised in Table 8.3.

Clinical diagnosis

First, it is important to consider the history of the patient's illness and the findings on physical examination, and to formulate a provisional diag­nosis and list the differential diagnoses.

1  Pancreatic disease may cause malabsorption of protein, fat or carbohydrate, due to deficiency of digestive enzymes.

2  Biliary disease may cause malabsorption of fat and fat-soluble vitamins, due to lack of bile acids.

3  Intestinal mucosal disease may affect digestion transport, or both, of many dietary constituents and reabsorption of bile acids. The effects may be general, or relatively specific.

4  Bacterial colonisation of the small intestine may cause a functional deficiency of bile acids, and so interfere with absorption of fats. It may also interfere with the digestion of protein or absorption of amino acids,  and  decrease  the  availability  of water-soluble vitamins.

 

 

Initial investigations

Microbiological    examination,     including    stool  microscopy and culture, should always be per­formed before biochemical tests are requested whenever an infectious cause of a GI disorder needs to be excluded.

A faecal specimen should be inspected; this may suggest that the patient has steatorrhoea. The specimen should also be tested for occult blood.

Preliminary biochemical investigations on blood specimens should include urea and electrolytes, albumin and other 'liver function tests', calcium and C-reactive protein. Preliminary haematological investigations (Hb, full blood count, vitamin B₁₂, folate and ferritin) should also be performed.

Further investigations

Radiology  (e.g.   barium  meal,   barium   enema) endoscopy (e.g. gastroscopy, duodenoscopy, ERCP, colonoscopy) and mucosal biopsy (e.g. duodenal biopsy) may be indicated. They may define the site of an anatomical abnormality, and are more reliable in this respect than most of the organ-directed biochemical tests considered in this chapter.

Coeliac serology Antiendomysial or anti tissue transglutaminase IgA.

Tests of pancreatic function, for example, faecal elastase.

Several other biochemical abnormalities may occur in association with intestinal malabsorption, and require appropriate investigation and treat­ment. These include:

1Vitamin deficiency.

2Defects in calcium absorption that may cause rickets or osteomalacia.

3Malabsorption  of iron This  may  cause  iron deficiency    anaemia.    Mixed    deficiencies    of vitamin B₁₂, folate and iron may also occur.

4       Malabsorption of protein Reduction in plasma albumin most often results, but hypogammaglobulinaemia may be marked.

Factitious diarrhoea is becoming increasingly common in tertiary referral centres and a high index of clinical suspicion may be necessary to prevent     extensive     needless     investigation. Possible laxative abuse should be investigated in a specialist laboratory by screening a random urine    sample   for   over-the-counter   laxatives including the colonic stimulants bisacodyl and senna. If possible, the urine sample should be collected at a time when the patient is known to have diarrhoea. It should be remembered that patients may use laxatives intermittently and that a single negative result does not exclude this

dignosis. Osmotic laxatives such as magnesium sulphate also may cause diarrhoea when used inappropriately and an elevated faecal osmotic gap may provide a clue to their use. The faecal osmotic  gap  is  calculated by  measuring  the sodium and potassium concentrations in faecal water and then doubling their sum to account for anions. This figure is then subtracted from an assumed osmolality of 290 mosm/kg which has been shown to give a close approximation to intracolonic osmolality.

Carcinoid tumours and the carcinoid syndrome

Carcinoid tumours arise in the gut or in tissues derived from the embryological foregut (e.g. thyroid, bronchus). The commonest sites are the terminal ileum and the ileocaecal region. The tumours produce vasoactive amines which, because of the venous drainage of the tumours, are usually carried directly to the liver and there inactivated. Symptoms are only likely to occur either when the tumour has metastasised to the liver, or when the tumour drains into the systemic circulation (e.g. bronchial adenoma of the carcinoid type).

Most carcinoid tumours secrete excessive amounts of 5-hydroxytryptamine (5-HT: serotonin), which is metabolised and excreted in urine as 5-hydroxyin-doleacetic acid (5-HIAA). Atypical carcinoid tumours secrete excessive amounts of 5-hydroxytryptophan (5-HTP) and relatively little 5-HT; they may also secrete histamine. Whereas only about 1% of dietary tryptophan is normally metabolised to 5-HTP, 5-HT and 5-HIAA, in the carcinoid syndrome, as much as 60 % of dietary tryptophan is metabolised along this hydroxyindole pathway.

The carcinoid syndrome is usually associated with tumours of the terminal ileum and extensive sec­ondary deposits in the liver. The main presenting features include flushing attacks, abdominal colic and diarrhoea, and dyspnoea, sometimes associ­ated with asthmatic attacks. Valvular disease of the heart is often present. Carcinoid tumours can give rise to severe hypoproteinaemia and oedema, even in the absence of cardiac complications. There may also be signs of niacin deficiency, due to major diversion of tryptophan metabolism away from the pathway leading to niacin production (p. 138). Some carcinoid tumours produce ACTH or ACTH-like peptides, and may cause Cushing's syndrome in the absence of the symptoms commonly associated with the carcinoid syndrome.

Biochemical investigation of 5-HT metabolism

Measurement of 5-HIAA excretion in a 24 h urine specimen is the most widely performed investigation; the output is usually greatly increased. Bananas and tomatoes contain large amounts of 5-HT; they should not be eaten the day before or during the urine collection.

Timing of urine collection If attacks are frequent, the time of starting the collection is unimportant. If attacks are less often than daily, the patient should be instructed to wait and begin the collec­tion when the next attack occurs.

GI hormones and Verner-Morrison syndrome

A number of GI hormones with various hormonal and local effects have been identified (Table 8.4). Excess amounts of these GI peptides are secreted by rare tumours. These tumours can often be identified by finding raised levels of the corre­sponding peptide in plasma. For example, in the Verner-Morrison syndrome, hypersecretion of vasoactiveintestinal peptide (VIP) causes severe watery diarrhoea and hypokalaemia.

 

PATOBIOCHEMISTRY OF LIVER

 

Liver’s functions:

It is responsible for the production of bile which is stored in the gallbladder and released when required for the digestion of fats.
The liver stores glucose in the form of glycogen which is converted back to glucose again when needed for energy.
It also plays an important role in the metabolism of protein and fats.

It stores the vitamins A, D, K, B₁₂ and folate and synthesizes blood clotting factors.
Another important role is as a detoxifier, breaking down or transforming substances like ammonia, metabolic waste, drugs, alcohol and chemicals, so that they can be excreted. These may also be referred to as "xenobiotic" chemicals.

Role of the liver in carbohydrate metabolism.

From intestine glucose pass into the liver, where most part of it undergone the phosphorillation. Glucose-6-phosphate formed in result of this reaction, which catalyzed by two enzymes – hexokinase and glucokinase. When level of glucose in blood of v. porta and in the hepatocytes is normal activity of glucokinase is low. After eating activity of this enzyme increase and blood level of glucose decrease because glucose-6-phosphate cannot pass through membrane.

Fructose and galactose also transformed into glucose-6-phosphate in the liver.

Glucose-6-phosphate is a key product of carbohydrates metabolism. In the liver this substance can metabolized into different ways depend of liver’s and whole organism’s necessity.

1. Synthesis of glycogen. Content in the liver – 70-100g. After eating amount of glycogen in the liver increase up to 150g. After 24 hours of starvation content of glycogen in the liver decreases to zero and glukoneogenesis started.

2. Glucose-6-phosphatase catalize dephosphoryllation of glucose-6-phosphate and free glucose formed. This enzyme is present in the liver, kidney and small intestine. This process keep normal level of glucose in the blood.

3. Excess of glucose-6-phosphate, which is not used for synthesis of glycogen and forming of free glucose, decomposites in glycolysis for pyruvate and for acetyl-CoA, which are used for fatty acids synthesis.

4. Glucose-6-phosphate decomposites for H₂O and CO₂, and free energy for hepatocytes is formed.

5. Part of glucose-6-phosphate oxidized in pentosophosphate cycle. This way of glucose decomposition supplyes reducted NADPH, which is necessary in fatty acid synthesis, cholesterol synthesis, and also pentosophosphates for nucleic acids. Near 1/3 of glucose in liver  is used for this pathway, another 2/3 – for glycolysis.

Role of the liver in lipid metabolism

 

In the liver all processes of lipid metabolism take place. Most important of them are following:

1. Lipogenesis (synthesis of fatty acids and lipids). Substrate for this process – acetyl-CoA, formed from glucose and amino acids, which are not used for another purposes. This process is very active when the person eats a lot of carbohydrates. Liver more active than another tissues synthesizes saturated and monounsaturated fatty acids. Fatty acids then used for synthesis of lipids, phospholipids, cholesterol ethers. Glycerol-3-phosphate, which is necessary for lipids synthesis, formed in liver in result of two processes: from free glycerol under influence of glycerolkinase, or in reducing of dioxiacetone phosphate under influence of glycerolphosphate dehydrogenase. Active form of fatty acids interact with glycerol-3-phosphate and phosphatidic acid formed, which used for synthesis of triacylglycerines and glycerophospholipids.

2. Liver play a central role in synthesis of cholesterol, because near 80 % of its amount is synthesized there. Biosynthesis of cholesterol is regulated by negative feedback regulation. When the level of cholesterol in the meal increases, synthesis in the liver decreases.  Cholesterol  is using in the body for building cell membranes, synthesis of steroid hormones and vitamin D. Excess of cholesterol leads out in the bile to the intestine. Another part of cholesterol is  used for bile acids synthesis.

3. Liver is a place of ketone bodies synthesis. These substances are formed from fatty acids after their oxidation, and from liver are transported to another tissues, first of all to the heart, muscle, kidney and brain. These substances are main source of energy for many tissues of our organism excepting liver in normal conditions (heart) and during starvation (brain).

Role of the liver in protein metabolism

 

Liver has full set of enzymes, which are necessary for amino acids metabolism. Amino acids from food are used in the liver for following pathways:

1. Protein synthesis.

2. Decomposition for the final products.

3. Transformation to the carbohydrates and lipids.

4. Interaction between amino acids.

5. Transformation to the different substances with amino group.

6. Release to the blood and transport to another organs and tissues.

The high speed of protein synthesis and decomposition is typical for the liver. Hepatocytes catch different protein from blood (from hemolysated RBC, denaturated plasma proteins, protein and peptide hormones) and decomposite them to the free amino acids which are used for new synthesis. When organism does not get necessary quantity of amino acids from food, liver synthesizes only high necessary proteins (enzymes, receptors).

Liver syntesizes 100 % of albumines, 90 % of α₁-globulines, 75 % of α₂-globulines, 50 % of β-globulines, blood clotting factors, fibrinogen, protein part of blood lipoproteins, such enzyme as cholinesterase. The speed of these processes is enough high, for example, liver synthesizes 12-16g of albumine per day.

Amino acids, which are not used for protein synthesis are transformed to another substances. Oxidative decomposition of amino acids is main source of energy for liver in normal conditions.

Liver can synthesize non-essential amino acids. Liver synthesizes purine and pyrimidine nucleotides.

Liver function tests

Most laboratories perform a standard group of tests (Table), which do not assess genuine liver function but are useful for:

1 . Detecting the presence of liver disease;

2 . Placing the liver disease in the appropriate broad diagnostic category. This then allows the selection of further, more expensive and time-consuming inves­tigations such as ultrasound, computed tomography (CT) scanning, endoscopy and liver biopsy;

3 . Following the progress of liver disease.

Routine liver function tests

 

Hepatic anion transport: bilirubin

Measurements of bilirubin in blood and urine are usually used to assess hepatic anion transport, although many other anions, including bile salts, are also transported by the liver. Understanding the mechanisms by which bilirubin is formed and removed is essential for the diagnosis of patients with jaundice or liver disease, since abnormal lev­els of bilirubin in blood can occur in patients in whom there is no liver disease.

Bilirubin production and metabolism

The body usually produces about 300 mg of bilirubin per day as a breakdown product of haem. About 80% arises from red cells with the remainder coming from red cell precursors destroyed in the bone marrov ('ineffective erythropoiesis'), and from other haem proteins such as myoglobin and the cytochromes.

After a life span of about 120 days the erythrocytes die. The dead erythrocytes are taken up by the phagocytes of the reticuloendothelial system of the body. About 7 gram of Hb is released daily from these phagocytosed erythrocytes. The Hb molecule is broken down into 3 parts:

(i) The protein (globin) part is utilized partly as such or along with other body proteins.

(ii) The iron is stored in the reticuloendothelial cells and is reused for the synthesis of Hb and other iron containing substances of the body.

(iii) The porphyrin part is converted to bile pigment, i.e. bilirubin which is excreted in bile.

The several stages, which are involved in the formation of bile pigment from Hb and the farther fate of this pigment, are given below:

1. Hemoglobin dissociates into heme and globin.

2. Heme in the presence of the enzyme, heme oxygenase, loses one molecule of CO and one atom of iron in Fe³⁺ form producing biliverdin. In this reaction, the porphyrin ring is cleaved by oxidation of the alpha methenyl bridge between pyrrole rings. The enzyme needs NADPH+H⁺ and O₂.

Biliverdin which is green in color is the first bile pigment to be produced; it is reduced to the yellow-colored bilirubin, the main bile pigment, by the enzyme biliverdin reductase requiring NADPH+H⁺.

Bilirubin is non-polar, lipid soluble but water insoluble. Bilirubin is a very toxic compound. For example, it is known to inhibit RNA and protein synthesis and carbohydrate metabolism in brain. Mitochondria appear to be especially sensitive to its effect. Bilirubin formed in reticuloendothelial cells then is associated with plasma protein albumin to protect cells from the toxic effects. As this bilirubin is in complex with plasma proteins, therefore it cannot pass into the glomerular filtrate in the kidney; thus it does not appear in urine, even when its level in the blood plasma is very high. However, being lipid soluble, it readily gets deposited in lipid-rich tissues specially the brain.

This bilirubin is called indirect bilirubin or free bilirubin or unconjugated bilirubin.

The detoxication of indirect bilirubin takes place in the membranes of endoplasmatic reticulum of hepatocytes. Here bilirubin interact with UDP-glucuronic acid and is converted to the water soluble form -bilirubin mono- and diglucoronids. Another name of bilirubin mono- and diglucoronids is conjugated bilirubin or direct bilirubin or bound bilirubin. This reaction is catalized by UDP-glucoroniltransferase.

Conjugated bilirubin is water soluble and is excreted by hepatocytes to the bile. Conjugated (bound) bilirubin undergoes degradation in the intestine through the action of intestinal microorganisms. Bilirubin is reduced and, mesobilirubin is formed. Then mesobilirubin is reduced again and mesobilinogen is formed. The reduction of mesobilinogen results in the formation of stercobilinogen (in a colon). Stercobilinogen is oxidized and the chief pigment (brown color) of feces stercobilin is formed. A part of mesobilinogen is reabsorbed by the mucous of intestine and via the vessels of vena porta system enter liver. In hepatocytes mesobilinogen is splitted to pyrol compounds which are excreted from the organism with bile. If the liver has undergone degeneration mesobilinogen enter the blood and is excreted by the kidneys. This mesobilinogen in urine is called urobilin, or true urobilin. Thus, true urobilin can be detected in urine only in liver parenchyma disease.

Another bile pigment that can be reabsorbed in intestine is stercobolinogen. Stercobolinogen is partially reabsorbed in the lower part of colon into the haemorroidal veins. From the blood stercobolinogen pass via the kidneys into the urine where it is oxidized to stercobilin. Another name of urine stercobilin is false urobilin.

As mentioned above, the conversion of bilirubin to mesobilirubin occurs under the influence of intestinal bacteria. These bacteria are killed or modified when broad-spectrum antibiotics are administered. The gut is sterile in the newborn babies. Under these circumstances, bilirubin is not-converted to urobilinogen, and the feces are colored yellow due to bilirubin. The feces may even become green because some bilirubin is reconverted to green-colored biliverdin by oxidation.

The total bilirubin content in the blood serum is 1,7-20,5 micromol/l, indirect (unconjugated) bilirubin content is 1,7-17,1 micromol/l and direct (conjugated) bilirubin content is 0,86-4,3 micromol/l.

 

 

Hepatocellular damage: aminotransferase measurements

Soluble cytoplasmic enzymes and, to a lesser extent, mitochondrial enzymes are released into plasma in hepatocellular damage. The measurement of the activity of ALT or AST in plasma provides a sensitive index of hepatocellular damage. Plasma ALT measurements are more liver-specific than AST. Cytoplasmic and mitochondrial isoenzymes of AST exist and in chronic hepatocellular disease (e.g. cirrhosis), serum AST tends to be increased to a greater extent than ALT. The aminotransferases are mainly located in the periportal hepatocytes, and they do not give a reliable indication of centrilobular liver damage. As with all tests based on the release of enzymes from damaged tissue, there is a lag period of some 24 h from the initiation of tissue damage to the first appearance of increased enzyme levels in the plasma.

AST- 8-40 U/L or (0,1-0,45 mmol/(hour´L))

ALT- 5-30 U/L or (0,1-0,68 mmol/(hour´L))

Cholestasis: alkaline phosphatase (ALP) and -γ-glutamyltransferase (GGT)

Some enzymes, such as ALP and GGT, are normally attached, or 'anchored', to the biliary canalicular and sinusoidal membranes of the hepatocyte. For this reason, ALP and GGT tend to be released into plasma in only small amounts following hepato­cellular damage. However, they are released in much greater amounts when there is cholestasis, since their synthesis is induced and they are ren­dered soluble - due, at least in part, to the presence of high hepatic concentrations of bile acids.

Changes in the activities of GGT and ALP often parallel each other in cholestatic liver disease. Plasma GGT has the advantage of being more liver-specific, as plasma ALP may also be increased due to release from bone in bone disease. However, alcohol and many drugs such as anticonvulsants may induce the expression of GGT without caus­ing cholestasis. An isolated increase in GGT should thus be interpreted with caution.

ALP – 40-125 U/L or  0,5-1,3 mmol/(hour´ L)

GGT – 6-45 U/L in male and 5-30 U/L in female.

 

Hepatic protein synthesis

The measurement of certain plasma proteins pro­vides an index of the liver's ability to synthesise protein.

Albumin

In chronic hepatocellular damage, there is impaired albumin synthesis with an accompanying fall in plasma [albumin]. Albumin measurements provide a fairly good index of the progress of chronic liver disease. In acute liver disease, however, there may be little or no reduction in plasma [albumin], as the biological half-life of albumin is about 20 days and the fractional clearance rate is therefore low. Factors other than impaired hepatic synthesis may lead to a decreased plasma [albumin]. These include loss of albumin into the extravascular compartment, ascites, increased degradation and poor nutritional status.

Ascites Increased portal venous pressure, a low plasma colloid oncotic pressure and Na⁺ retention due to secondary hyperaldosteronism combine to cause ascites in cirrhotic patients. This often devel­ops when plasma [albumin] falls below 30 g/L.

Coagulation factors

In liver disease, the synthesis of prothrombin and other clotting factors is diminished, leading to an increased PT. This may be one of the earliest abnormalities seen in patients with hepatocellular damage, since prothrombin has a short half-life (approximately 6 h). The PT is often expressed as a ratio to a control value (the international nor­malised ratio, INR).

Deficiency of fat-soluble vitamin K, due to failure of absorption of lipids, may also cause a prolonged PT. In vitamin K deficiency, the coagulation defect can often be corrected by parenteral administra­tion of vitamin K, but this has no effect in patients with hepatocellular damage.

Immunoglobulins

 

Plasma Ig measurements are of little value in liver disease because the changes are of low specificity. In most types of cirrhosis, plasma [IgA] is often increased, while in primary biliary cirrhosis plasma [IgM] increases greatly. In chronic active hepatitis, plasma [IgG] tends to be most increased.

Serological tests

Anti-mitochondrial antibodies are present in over 95% of patients with primary biliary cirrhosis, and anti-smooth muscle and anti-nuclear factor antibodies are found in about 50 % of patients with chronic active hepatitis. Viral antigens and antibody measurements are also important in detecting infective causes of liver disease.

Marker of fibrosis

A variety of markers have been described that may be of help in the assessment of hepatic fibrosis. Procollagen type III terminal peptide and hyaluronic acid (hyluronin) are the most commonly used tests.

Other liver function tests

A number of liver function tests have been described that give an indication of the functional liver mass. These tests are not often used but include the

aminopyrine breath tests, the galactose elimination test and the monoethylglycinexylidide (MEGX) test.

Disordered metabolism

Patients with severe liver disease may have

1. significant decreases in plasma [urea], due to fail­ure of the liver to convert amino acids and NH₃ to urea.

2. hypoglycaemia due to impaired gluconeogenesis or glycogen breakdown, or both;

3. raised concentrations of all the plasma lipid fractions, if cholestasis is present. An abnormal lipoprotein that contains high concentrations of phospholipid, lipoprotein X, is present in plasma in nearly all the cases of cholestasis.

The place of chemical tests in the diagnosis of liver disease

The jaundiced patient

Jaundice or icterus is the orange-yellow discoloration of body tissues which is best seen in the skin and conjunctivae.

 Jaundice   is   due   to   hyperbilirubinaemia   and becomes clinically apparent when the plasma bilirubin exceeds about 40-50 μmol/l.

 

 

Pre-hepatic hyperbilirubinaemia:

 This is due to overproduction of bilirubin. It occurs in:

·        haemolytic anemia

·        haemolytic disease of newborn, due to rhesus incompatibility

·        ineffective erythropoiesis (e.g. pernicious anemia)

·        Rhabdomyolysis

Hepatocellular hyperbilirubinaemia

This can arise from:

Hepatocellular damage caused by infective agents, drugs and toxins

Cirrhosis

Low activity of  bilirubin UDP-glucuronyltransferase in congenital deficiency, premature infants or competitive inhibition of the enzyme by drugs (novobiocin).

Cholestatic hyperbilirubinaemia

Cholestasis may be intrahepatic or extrahepatic. In both, there is conjugated hyperbilirubinaemia and bilirubinuria.

Cholestasis commonly occurs in:

·        Acute hepatocellular damage (e.g. due to infectious hepatitis)

·        Cirrhosis

·        Intrahepatic carcinoma (most commonly secondary)

·        Primary biliary cirrhosis

·        Drugs (e.g. methyltestosterone, phenothiazines).

Extrahepatic cholestasis is most often due to:

·        gallstones

·        carcinoma of the head of the pancreas

·        carcinoma of the biliary tree

·        bile duct compression from other causes.

Hemolytic jaundice is characterized by

1.     Increase mainly of unconjugated bilirubin in the blood serum.

2.     Increased excretion of urobilinogen with urine.

3.     Dark brown colour of feces due to high content of stercobilinogen.

Hepatic jaundice is characterized by

1.Increased levels of conjugated and unconjugated bilirubin in serum.

2.Dark coloured urine due to the excessive excretion of bilirubin and urobilinogen.

3.Pale, clay coloured stools due to the absence of  stercobilinogen.

4.Increased activities of alanine and aspartate transaminases.

 

 

Obstructive (post hepatic ) jaundice is characterized by

1.Increased concentration mainly of conjugated bilirubin in serum.

2.Dark coloured urine due to elevated excretion of bilirubin and clay coloured feces due to absence of stercobilinogen.

 

The congenital hyperbilirubinaemias

These are all due to inherited defects in the mechanism of bilirubin transport and metabolism.

Gilbert's syndrome

This familial autosomal dominant trait is probably present in 2-3% of men; it is 2-7 times more common in men than women. The unconjugated hyperbilirubinaemia is usually asymptomatic, and plasma [bilirubin] fluctuates, higher levels tending to occur during intercurrent illness. Most patients have a plasma [bilirubin] less than 50 μmol/L, but higher levels are not uncommon. Other com­monly performed tests of 'liver function' are normal, and there is no bilirubinuria.

Gilbert's syndrome is caused by decreased expres­sion of bilirubin UDP-glucuronyltransferase 1A1, due to a mutation in the promoter portion of the gene.

Gilbert's syndrome can most easily be differenti­ated from the mild degree of hyperbilirubinaemia in haemolytic anaemia by haematological investi­gations. Confirmatory tests for Gilbert's syndrome include monitoring the effects on plasma [bilirubin] of a reduced energy intake (1.67 MJ/day; 400 kcal/day), particularly a reduction in the intake of lipids, for 72 h. This results in at least a doubling of plasma [unconjugated bilirubin] in patients with Gilbert's syndrome, whereas in normal individuals it does not rise above 25 μmol/L. Diagnosis by genotyping is possible but is rarely performed at present although it is likely to be increasingly performed in the future.

Fasting plasma [bile acids] are normal in Gilbert's syndrome, but raised in hyperbilirubinaemia due to liver disease.

Crigler-Najjar syndrome

This rare condition, due to low activity of biliru­bin UDP-glucuronyltransferase, gives rise to severe hyperbilirubinaemia in neonates, leading to kernicterus and often to early death.

Dubin-Johnson syndrome and Rotor's syndrome

These rare disorders are characterised by a benign conjugated hyperbilirubinaemia, accompanied by bilirubinuria. In both, there is a defect in the transfer of conjugated bilirubin into the biliary canaliculus. Urinary coproporphyrins are normal in patients with Dubin-Johnson syndrome, but increased in Rotor's syndrome.

Acute hepatitis

This is usually caused by viruses (hepatitis A, B, C, D and E, cytomegalovirus or Epstein-Barr). Toxins such as ethanol and paracetamol can also damage the liver. There is often a pre-icteric phase when increases in ALT and AST activities and in urobilinogen in urine occur. By the time clinical jaun­dice appears, plasma ALT and AST activities are usually more than 6 times, and occasionally more than 100 times, the upper reference value. The stools may be very pale, due to impaired biliary excretion of bilirubin, and urobilinogen then disappears more or less completely from the urine. ALP activity is usually only slightly increased, up

to about twice the upper reference value, but it may be considerably raised in cases (relatively uncommon) in which there is a marked cholestatic element, as occurs in acute alcoholic hepatitis. Acute viral hepatitis usually resolves quickly and chemical indices of abnormality revert to normal within a few weeks.

Poisoning and drugs

Findings similar to those in acute viral hepatis are observed in patients with hepatocellular toxicity due to drugs (e.g. paracetamol overdose, halothane jaundice, carbon tetrachloride poisoning). Drugs such as chlorpromazine may produce cholestasis, with increased plasma ALT and GGT, while phenytoin, barbiturates and ethanol induce GGT synthesis without necessarilv causing liver damage. Certain herbal remedies and recreational drugs such as ecstasy may also induce liver damage.

Acute liver failure

This rare condition is usually caused by paraceta­mol poisoning or hepatitis virus and the prognosis is often poor. It is accompanied by major meta­bolic disturbances including hyponatraemia, hypocalcaemia, hypoglycaemia and lactic acidosis often masked by respiratory alkalosis. The levels of the aminotransferases do not correlate well with the severity of the disease.

Chronic hepatitis

Hepatic inflammation that persists for more than 6 months is regarded as 'chronic hepatitis'. It may be due to chronic infection with hepatitis virus, alcohol abuse or be autoimmune in origin. Usually such patients have an isolated elevation in serum aminotransferase unless the disease has progressed to cirrhosis. Autoimmune hepatitis is frequently treated with azathioprine. The thera­peutic action of the azathioprine depends on the production of active metabolites. Toxicity can occur in patients who have low activities of the enzyme thiopurine methyl transferase (TPMT).

Cholestatic liver disease

Both extrahepatic (e.g. gallstones) and intrahepatic (e.g. tumours, certain drugs) causes of obstruc­tion cause cholestasis. The distinction between the two is often clinically important from the point of view of further investigation and treat­ment, but it can rarely be made by chemical tests.

Plasma [bilirubin] is often greatly increased, and there is marked bilirubinuria; urobilinogen often becomes undetectable in urine. Plasma ALP and GGT activities are considerably increased, often to more than three times the upper reference values, but plasma ALT and AST activities are usually only moderately raised. In long-standing cholestatic jaundice, hepatic protein synthesis may be impaired and plasma ALP activity may start to fall 15 a result, and even return to normal; this emphasises the importance of performing a baseline set of investigations as early as possible in patients with liver disease.

Plasma ALP and GGT activities may be markedly increased in patients with partial biliary obstruc­tion, due to local obstruction in one of the smaller biliary ducts, such as often occurs in both primary and secondary carcinoma of the liver. Partial bil­iary obstruction may have little or no effect on the capacity of the liver to excrete bilirubin, so there may be no evidence of jaundice in these patients, at least initially; bilirubin excretion in the other parts of the liver may be capable of fully compen­sating for the sector affected by the local biliary obstruction.

Chemical features that may help to distinguish cholestasis from hepatocellular damage are sum­marised in Table 7.3. These are 'typical' findings, and many cases do not follow these patterns exactly. The distinction between intrahepatic and extrahepatic cholestasis is usually made by radio­logical investigations - for example, endoscopic retrograde cholangiopancreatography (ERCP), ultrasound or CT scanning - or by liver biopsy.

Infiltrations of the liver

The liver parenchyma may be progressively disor­ganised and destroyed in patients with primary or secondary carcinoma, lymphoma, amyloidosis, reticuloses, tuberculosis, sarcoidosis and abscesses. These diseases often lead to partial biliary obstruc­tion, with the associated chemical changes described above. Plasma [α₁-fetoprotein] (AFP) is often greatly increased in hepatoma  but it can be moder­ately increased in chronic hepatitis and cirrhosis. Plasma AFP measurements are also useful for moni­toring patients who are at increased risk of develop­ing hepatoma. Patients with liver tumours may often have elevated ALP and GGT as the only abnor­mality due to localised obstruction.

Cirrhosis of the liver

Alcoholism, viral hepatitis, autoimmune disease and prolonged cholestasis are the most frequent known causes of cirrhosis in Britain, although in half the cases no obvious cause is found. Less often, cirrhosis is associated with metabolic disorders such as Wilson's disease, cystic fibrosis, API deficiency, haemochromatosis, or galactosaemia.

Mild cirrhosis.  In mild cases, no clinical abnormali­ties may be apparent, due to the reserve functional capacity of the liver. Plasma GGT measurements provide a sensitive means of detecting mild cir­rhosis, but most heavy drinkers (many of whom do not have cirrhosis of the liver) have raised plasma GGT activities; these usually fall within 2 months of stopping drinking. Marked abnor­malities in liver function tests are rarely present.

Severe cirrhosis The following clinical features may occur, either alone or in combination: haematemesis, ascites and acute hepatic decompensa­tion, often fatal. Jaundice may develop, plasma [albumin] falls and the PT becomes abnormal. Clinical deterioration accompanied by prolonged PT, a generalised amino aciduria, increased plasma [NH₃] and reduced plasma [urea] may herald the development of acute hepatic failure.

Hyaluronan (also known as hyaluronic acid) is a glucosaminoglycan synthesised by the mesenchymal cells and degraded by hepatic sinusoidal endothelial cells by a specific receptor-mediated process. Elevated levels are associated with sinusoidal capilliarisation that is seen in cirrhosis. Hyaluronan levels are significantly higher in patients with liver cirrhosis compared with hepatic fibrosis, chronic hepatitis and fatty liver. Measurement of fasting serum hyaluronan can reliably differentiate cirrhotic from non-cirrhotic liver disease and can be regarded as a useful test in the diagnosis of liver cirrhosis, particularly when a liver biopsy is contraindicated. There appears to be no significant difference in hyaluronan levels between cirrhosis caused by different aetiologies but hyaluronan levels are increased proportionally to the severity of cirrhosis.

Copper in liver disease

The liver is the principal organ involved in copper metabolism. The amount it contains is main­tained at normal levels by excretion of copper in bile and by incorporation into ceruloplasmin. The liver's copper content is increased in Wilson's disease, primary biliary cirrhosis, pro­longed extrahepatic cholestasis, and intrahepatic bile duct atresia in the neonate.

Wilson's disease (hepatolenticular degeneration) is a rare, hereditary, autosomal recessive disorder with a prevalence of about 1 in 30 000. The defective gene encodes a protein involved in the hepatobiliary excretion and renal reabsorption of copper. Copper is deposited in many tissues, including the liver, brain, eyes and kidney. Symptoms are mainly due to liver disease and to degenerative changes in the basal ganglia. Plasma [ceruloplas­min] is nearly always low, but it is not clear how this relates to the aetiology of Wilson's disease.

The diagnosis may be suspected from the family history or on clinical grounds, such as liver disease in patients less than 20 years old, or characteristic neurological disease. Kayser-Fleischer rings, due to the deposition of copper in the cornea, can be detected in most patients. The following chemical tests may be valuable:

·        Plasma [ceruloplasmin] This is usually less than 200 mg/L (reference range 250-450 mg/L).

·        Plasma   [copper] This   is   usually   less   than 12 μmol/L (reference range 12-26 (μmol/L).

·        Urinary copper output This is always more than 1. 0 μmol//24 h (normally below 0.5 μmol/24 h).

·        Liver [copper] is always greater than 250 μg/g dry weight (reference range 50-250 μg/g dry weight).
This is the most sensitive test, but it involves liver biopsy.

These tests are not 100 % specific for Wilson's disease. For example, plasma [ceruloplasmin] may occasionally be low in severe cirrhosis, and uri­nary copper output and liver [copper] may be raised in biliary cirrhosis. However, urinary copper output is valuable for case-finding among rela­tives, since a normal result virtually excludes Wilson's disease.

Abnormalities of other chemical tests are often present in Wilson's disease. There is usually evidence of renal tubular damage, with a generalised

(overflow) amino aciduria, glycosuria and phosphaturia and, in advanced cases, renal tubular acidosis.

Ascites

Liver disease is the commonest cause of ascites. If a diagnostic paracentesis is performed, the appearance of the fluid (blood-stained, bile-stained, milky,  etc.)  should be noted, and fluid [total protein] should be determined.

Transudates and exudates

Ascites with a fluid [protein] less than 30 g/L is called a transudate. It is usually associated with non-infective causes such as uncomplicated cirrhosis, in which there is a combination of back-pressure effects and low plasma [albumin]. However, fluid [protein] may be greater in some of these patients, and 30 g/L is not a reliable diagnostic cut-off point.

Ascites with a fluid [protein] much in excess of 30 g/L is called an exudate. It usually indicates the presence of infective conditions such as tubercu­lous peritonitis, or malignant disease or pancreatic disease. If pancreatic disease is thought to be the cause, fluid amylase activity should be measured; a serosanguinous fluid with a high amylase activ­ity will help to confirm the diagnosis. If hepatoma is suspected, plasma and ascitic fluid [AFP] may both be considerably increased.