CHAPTER 1
Introduction
1.1 Importance of Trace Elements in Food
1.1.1 Essential Trace Elements
Food and beverages ingested by humans represent a potentially proficient pathway of exposure to toxic and nutritionally important minor and trace elements. Many mineral elements occur in living tissues, food and diets in such small amounts that they are frequently described as "traces" and the phrase "trace elements" arose to describe them. At the present time, less than one-third of the 90 naturally occurring elements are known to be essential for life.
The bulk of human body is composed of six major elements; oxygen, carbon, hydrogen, nitrogen, calcium and phosphorus and six minor elements; sulfur, potassium, sodium, chlorine, magnesium and silicon. The total percentage of minor and major elements in total body weight is 98.8 (Table 1.1). If six noble gases are excluded as unlikely to have a physiological function, 71 elements of the periodic system remain, and because of their low concentration in living matter, are termed the "trace elements".
The concentration of major and minor elements in living tissues can be expressed in grams per kilogram. On the other hand, the concentration of trace elements in living tissues varies between 0.01 and 100 mg kg-1 (Table 1.2). It may not be appropriate to classify them as essential or toxic elements. It is logically wrong to establish a category of "toxic" elements, because any element may be potentially toxic and this property is but a function of concentrations to which humans are exposed. Essentiality of the trace elements is established when a further reduction below the range of tolerable levels, better known as "range of safe and adequate intakes", results in a consistent and reproducible impairment of a physiological function.
These considerations suggest a logical classification of the 71 trace elements into those, with proven essentiality and the rest for which essentiality is "presently not known". This classification leaves room for the possibility that future research will include additional elements as essential. Each of the two categories can be subdivided according to their practical importance under given conditions; e.g., local, regional or national imbalance in the environment, industrial emissions or dietary habits. Some essential elements may not be of any nutritional concern at all, as in the case of magnesium in human nutrition, since it is in sufficiently high levels; others, such as selenium, may have the highest regional importance because of deficiency in one area and toxicity in another.
In recent years, there has been an increase in the realization of the importance of the role of trace elements in biological systems. The study of life processes shows that many vital functions are dependent on the presence of a specific trace element. Because of that, trace elements are one of the important nutrient factors for the growth and maintenance of human and animal life.
Food only, excluding intakes from water and air, normally supplies a major proportion of the total daily trace element intake by humans. Since the late 1950s, concerns over the introduction of trace elements and many other components into the environment as a result of human activities have greatly increased. Besides soil and water, food is also contaminated with trace metals by the introduction of mechanized farming, ever increasing use of chemicals, sprays, preservatives, food processing and canning. In order to get the minimum adverse impact, it is important to measure and continuously monitor their levels in various food items, total diet, water and inhaled air.
The concentrations of trace elements in food give important information about dietary habits of special group, health situation of individuals and origins of elements. Therefore, it is important to determine the daily dietary intake of trace elements, their concentrations and sources.
Recent developments of trace element research in the area of nutrition have led to a need to accurately and precisely determine the content of these micronutrients in food. In the past several decades, the analytical chemistry community has made great advances in improvement in sensitivity, selectivity and accuracy of analytical methodology.
In this book, we will present experimental techniques for the collection, preparation and determination of trace elements in food. All the modern techniques will be discussed in some detail so that it will be useful for both researcher and technical staff who are working in this area.
1.1.2 Classification of Trace Elements
The simplest definition of trace essential element is that it is required in small amount for the maintenance of life; its absence results in death or a severe malfunction of the organism.
All major and minor elements are important; besides that, some of the trace elements e.g; Cr, Fe, Co, Cu, Zn, Se, Mo and I are essential trace elements; and some of them; Mn, Si, Ni, B, V, and Sn are probably essential trace elements; and further some of them F, As, Cd, Pb, Al and Hg are considered potentially toxic, some possibly essential elements for animal and human life. Actually all essential elements may also be toxic in animals and humans if ingested at sufficiently high levels and for a long enough period (Fig.1.1). The above elements will be discussed in detail in Chapter 13.
Essential trace elements are required by man in amounts ranging from 50 µg day-1 to 20 mg day-1. The organism can neither grow nor complete its life cycle without the element in question. The element should have a direct influence on the organism and be involved in its metabolism. The effect of the essential element cannot be wholly replaced by any other element.
The bioavailibilities of the essential elements depend on their chemical form, the compositions of diet and health situation of the individuals. Thus, establishment of the optimum daily requirements and determination of actual daily intake of essential elements are important problems of trace element in nutrition.
The essential trace elements provide a classical example of required nutrients as described by Bertnard as early as 1911. An organism may go through several stages as the concentration of essential nutrient progresses from deficiency to excess. In absolute deficiency, death may result, with limited intake; the organism survives but may show marginal insufficiency. With increasing nutrient, a plateau representing the optimal function is reached. As the nutrient is given in excess, first marginal toxicity then mortal toxicity are attained while this curve may vary quantitatively for each essential nutrient, the basic pattern holds for virtually all the essential trace elements. This is illustrated in Figure 1.2 for selenium. There is barely a fourfold range between intake per day for survival and that for the appearance of toxic effects.
1.1.3 Discovery of Essential Trace Elements
The study of the discovery of essential trace elements has been outlined by Schrauzer. The treatment of anaemia with iron and the association of iodine deficiency with goiter marked these as the only two essential trace elements recognized for animals before the twentieth century. In the twentieth century, there were two major periods of activity in biological trace element research. In the early classical period, 1925–1956, the essentiality of copper, zinc, cobalt, manganese and molybdenum in animals was discovered. A more active modern period, 1957–1980, dominated by the late Klav Schwarz, was marked by the experimental induction of trace element deficiencies. These efforts have resulted in evidence supporting the essentiality of selenium, chromium, tin, vanadium, fluorine, silicon, nickel, lead, cadmium, arsenic and most recently lithium.
1.1.4 Functions of Trace Elements
Most of the trace elements serve a variety of functions, depending upon their chemical form or combination and their location in the body tissues and fluids.
Minor and trace elements serve in two general roles. The first one is their function as structural material. Iron is part of the structure of the oxygen-carrying protein, haemoglobin, in the red blood cells; calcium, phosphorus and other elements constitute a significant part of the mass of teeth and bones; and sodium, potassium, phosphate, sulfate, chloride and many other elements are important constituents of the fluids, both inside and outside all the body cells.
The second general role of trace elements is their function in regulating numerous biological activities. Calcium in minute concentrations is necessary for normal blood clotting; magnesium stimulates the activity of many enzymes and a number of trace elements control the contraction of muscle and the transmission of impulses by nerve cells. Table 1.3 lists the macrominerals and trace elements known to be essential in human nutrition and their functions.
1.2 Trace Element Studies
The study of trace element contents in food, environmental and biological samples has attracted worldwide interest, and a lot of papers are published in this field. Since early 1970s, there has been an increasing interest in the levels of several elements in composite diet and individual food items such as honey, meat, milk, wheat, water, fish and vegetables. Also a great deal of research has been undertaken on the concentration of essential trace elements in biological materials such as fluids and tissues. Attempts have been made in recent years to understand the role of trace elements in biological system, particularly in human metabolism.
The results obtained by the analyses of the trace elements in foods may not show the exact elemental values taken by human daily that may be lost due to contamination during washing, cooking and eating procedures.
CHAPTER 2
Statistical Evaluation of Data
2.1 Introduction
Statistics is concerned with the organization, analysis and interpretation of numerical data. Since many results are obtained during trace element analysis of food and diet, statistical evaluation of data is most important. An analyst should know how to describe the results of the measurements, understand the statistics used in evaluation of the data, has to interpret the various types of data and make the basic statistical computations. The statistical evaluations are mostly used for
(1) measuring the central tendency,
(2) measuring the variability and
(3) measuring the relationship between different measurements.
The first two tasks provide a convenient means of analysing and describing a single set of data, and the last one can be used to indicate the agreement between data from different sources or different data sets.
2.2 Analytical Errors
2.2.1 Accuracy and Precision
In most chemical analyses, the true value is not known and error arises from the method, instruments, etc. Therefore, statistical analysis has to be used to determine the errors and to obtain the reasonable expression of results. In calculations, it is necessary to make a distinction between the exact and approximate values. Most of the results are approximate, since an interval and not exact points on some scale represent them. For example, if a food sample weighed for trace element analysis is 0.056 g, then it is expected that its value will be between 0.055 and 0.057 g. The deviation from the exact value is expressed in terms of accuracy, which can be defined as the correctness of a measurement or the nearness of a measurement to the true value. If, for example, a true value is ??and experimental value x i, then the difference between the two values is the absolute error,
absolute error = xi - μ (2.1)
The error is a measure of the accuracy of that determination. In practice, the error is often expressed in terms of percent relative error, percent relative error
percent relative error = (x)i x 100/μ (2.2)
Thus, the accuracy of a measurement is often expressed in terms of percent relative error.
In most analysis, the actual value of measurement is not known with any degree of exactness. However, the agreement between the repeated measurements should still be satisfactory. This is expressed in terms of precision, which can be defined as the measure of the reproducibility of a measurement.
Accuracy and precision are different characteristics of a set of measurements, and they should be correctly interpreted. Accuracy expresses the correctness, and precision is the reproducibility of a measurement. A good precision does not mean a good accuracy, because it is possible to repeat the same error systematically for a measurement. However, for an acceptable measurement, both the precision and accuracy should be reasonably good.
2.2.2 Determinate and Indeterminate Errors
Absolute error is the difference between a measured value and the true value Equation (2.1). In an experiment the errors may be classified as determinate (systematic) and indeterminate (random).
Determinate errors have definite values with positive or negative directions; their sources can be found and the error can often be corrected. Therefore, they have a rather constant nature from one measurement to another. The most common determinate errors are due to improper calibration of instruments and use of instruments by an inexperienced or careless person. Also a colour-blind person cannot accurately differentiate between colours during a titration where visual indicators are used. If the method chosen is not suitable for the analysis, a serious error will be obtained which cannot be corrected easily. For example, if a gravimetric method is used for an analyte, which does not have a small solubility product, the results will be inaccurate.
Indeterminate errors are experimental errors, as a result of small differences in replicated results. This type of error is not systematic and cannot be corrected. The most important source of random errors may be the result of unknown in homogeneity of the sample, impurities in the sample, instrumental fluctuations, imperfections in the experimental technique and fluctuation in experimental conditions, such as temperature, conductivity, electrical voltage.
In almost every experiment there may be some error, which have to be corrected either directly or statistically. The determinate error usually gives the degree of accuracy, whereas indeterminate error gives the degree of precision. The accuracy is dictated mostly by determinate errors where the precision is a function of indeterminate errors.
2.2.3 Significant Figures
Significant figures are the digits in a number including all the digits known with certainty plus the first one of the estimated digits. In calculations, the numbers are usually rounded off to a lower number of significant figures as limited by the input value. The number in question can be an integer or fractional number. Thus, when a number is rounded off to have smaller number of significant figures, and if the disappearing digit is 0–4, the last digit of the result remains the same; when the disappearing digit is 6–9, the last digit is increased by 1. When the disappearing digit is 5, and if the last digit is an odd number, it is increased by 1; if it is an even number, it will be kept as it is. For example, when rounding the numbers down to three significant figures, 6.632, 6.638, 6.635 and 6.645 become 6.63, 6.64, 6.64 and 6.64, respectively.
The uncertainties in most of the analytical measurements depend on the instruments used. For example, an analytical balance, which has a precision of 0.1 g, can read a value such as 4.4 [+ or -] 0.1 g, where a balance with a precision of 0.1 mg will read 4.4615 [+ or -] 0.0001 g. Large numbers are expressed in powers of 10 to make the calculation simpler. However, the significant figures have to be considered in this form. For example, the weight 1245 mg can be written as 1.245x103 mg, but 3870.0 mg has to be written as 3.8700x103 mg.
In calculations, the significant figures have to be considered to obtain realistic results. In addition and subtraction type calculations, the number of significant figures is determined by the location of the decimal point and can be seen by visual inspection. Here, the input value with the smallest number of digits after the decimal point is limiting. However, it is best to retain all the digits until the arithmetic operation ends; the result will then be rounded. For example,
362.2
18.225
+5.3062
385.7312
Since the limiting number is 362.2, the result should be rounded to 385.7.
In multiplication or divisions, the number of significant figures in the resulting value will have the number of significant figures, which is limited by the input value with the lowest number of significant figures. If calculation contains both exact and approximate numbers, the number of significant figures in the result is determined by the number of significant figures in the approximated number. Therefore the molecular weight of N2 is 2x14.0067 = 28.0134 but not 3x101.
In log terms, the result should have a number of significant figures, which equals to the number of digits before the exponential plus the number of digits appearing as the power of 10. For example, the pH of 3.4x109 M H+ is 8.47. The first digit (8) comes from exponent (10-9) and fraction, 0.47 from two significant figures of 3.4.
2.3 Mean, Median, Mode, Range and Mean Deviation
The prediction of the best value from experimental results can be done by calculating the central tendency of the set of results. There are four types of central tendencies in common use: mean, median, mode and range.
