Biochemistry of Nutrition Topic 12 Nutrition Methodology PDF

Biochemistry of Nutrition BIOC1305  Topic 12 Nutrition Research Methodology  Statistical analysis and experimental design In all areas of research, statistical analysis of results and data plays a pivotal role. This chapter is intended to give students some of the very b...

Biochemistry of Nutrition BIOC1305  Topic 12 Nutrition Research Methodology  Statistical analysis and experimental design In all areas of research, statistical analysis of results and data plays a pivotal role. This chapter is intended to give students some of the very basic concepts of statistics as it relates to research methodology Validity Validity describes the degree to which the inference drawn from a study is warranted when account is taken of the study methods, the representativeness of the study sample and the nature of its source population. Validity can be divided into internal validity and external validity. Internal validity refers to the subjects sampled. External validity refers to the extension of the findings from the sample to a target population. Accuracy It describes the extent to which a measurement is close to the true value, and it is commonly estimated as the difference between the reported result and the actual value. Reliability It refers to the consistency or repeatability of a measure. Reliability does not imply validity. A reliable measure is measuring something consistently but not necessarily estimating its true value. If a measurement error occurs in two separate measurements with the same magnitude and direction, this measurement may be fully reliable but invalid. Precision Precision is described as the quality of being sharply defined In a more restricted statistical sense, precision refers to reducing random error. It can be improved by increasing the size of a study or using a design with higher efficiency.  Accuracy is a term used to describe the extent to which a measurement is close to the true value, and it is commonly estimated as the difference between the reported result and the actual value 5 Sensitivity and specificity Measures of sensitivity and specificity relate to the validity of a value. Sensitivity is the proportion of subjects with the condition who are correctly classified as having the condition. Specificity is the proportion of persons without the condition who are correctly classified as being free of the condition by the test or criteria. Sensitivity reflects the proportion of affected individuals who test positive, while specificity refers to the proportion of nonaffected individuals who test negative 6  Importance of generating and testing hypothesis generating a testable working hypothesis is the first step towards conducting original research. Such research may prove or disprove the proposed hypothesis. Case reports, case series, online surveys and other observational studies, clinical trials, and narrative reviews help to generate hypotheses. A hypothesis should be tested by ethically sound experiments with meaningful ethical and clinical implications. The coronavirus disease 2019 pandemic has brought into sharp focus numerous hypotheses, some of which were proven (e.g. effectiveness of corticosteroids in those with hypoxia) while others were disproven (e.g. ineffectiveness of hydroxychloroquine and ivermectin)  Sample size calculation One of the pivotal aspects of planning a clinical study is the calculation of the sample size. It is naturally neither practical nor feasible to study the whole population in any study. Hence, a set of participants is selected from the population, which is less in number (size) but adequately represents the population from which it is drawn so that true inferences about the population can be made from the results obtained. This set of individuals is known as the “sample.” In a statistical context, the “population” is defined as the complete set of people (e.g., Indians), the “target population” is a subset of individuals with specific clinical and demographic characteristics in whom you want to study your intervention (e.g., males, between ages 45 and 60, with blood pressure between 140 mmHg systolic and 90 mmHg diastolic), and “sample” is a further subset of the target population which we would like to include in the study. Thus a “sample” is a portion, piece, or segment that is representative of a whole.  In vitro studies In vitro studies represent part of the reductionist approach in nutrition research. The range of techniques used is large: Chemical analysis studies provide data on nutrient and non-nutrient content of foods. Digestibility techniques, in which a substrate is exposed to enzymes capable of digesting the substrate, help to refine the gross chemical analytical data to predict nutritional potential. Intact organs such as the liver of experimental animals can be used in studies such as perfused organ studies. In such studies, the investigator can control the composition of material entering an isolated organ and examine the output.  Sections of organs can also be used, such as the everted gut sac technique. A small section of the intestine is turned inside out and placed in a solution containing some test material Another approach is the construction of mechanical models that mimic an organ, usually the gut (in nutrition research). Many of these models successfully predict what is observed in vivo and have advantages such as cost and flexibility in altering the experimental conditions with great precision.  Animal models in nutrition research Whole animal systems have been used in measuring the utilization, function, and fate of nutrients. There are many reasons for choosing an animal study over a human study. We can and do subject animals to experimental conditions that we would ethically not be allowed to apply to humans. For example, to study how a nutrient influences the scale and histopathology of atherosclerosis, animal studies are needed. Just as studies with humans are governed by the rules of ethics committees, so too are studies with animals.  In general, the use of animals as models for human nutrition research can be examined from three aspects: the animal model the experimental diet and its delivery the experimental techniques available The animal model Many species have been used in the study of nutrition. Many are pure-bred strains such as the Wistar rat, the Charles River mouse, or the New Zealand white rabbit. Some animal models have been specially selected to exhibit particular traits, making them very useful models for research. The Wattanable rabbit has defective low-density lipoprotein (LDL) receptor function, making this animal model very useful for studying the role of diet in influencing LDL receptor mediated arterial disease.  The ob/ob mouse develops gross obesity because of an alteration in a genetic profile (leptin synthesis). In recent times there has been a rise in the use of transgenic animal models that have been produced through advanced molecular genetic techniques. In such models, specific genes can be inserted or deleted to fulfill specific functions. For example, the peroxisome proliferator-activated receptor- alpha (PPAR-) is not expressed in one knockout mouse model, giving rise to fat accumulation. Another example of a transgenic mouse presents an overexpression of the Cu/Zn-superoxide dismutase enzyme.  The experimental diet and its delivery The nature of the diet and its mode of delivery are centrally important in understanding the role of animal models in human nutrition issues. There are several types of diets offered to laboratory animals. Commercially available diets made to internationally accepted nutritional norms are often referred to as chow diets or laboratory chow. For the vast majority of laboratory animals in studies where nutrient intake is not the central area of interest, such chow diets are used. However, when nutrition is the area of research, special diets will almost always have to be formulated. The type of diet that needs to be formulated will depend on the nature of the research question.  In ad libitum feeding the animals have free access to food; in controlled feeding animals are offered a limited amount of food (restricted feeding) or receive as much food as can be fed to them (forced feeding). There are many reasons why pair feeding is critically important. An experiment may seek to examine how a new protein source, rich in some nutrient of interest, influences some aspect of metabolism. Let us consider a compound in the protein source that may reduce blood LDL cholesterol. A control diet is constructed based on casein. In the experimental diets, this casein is replaced on an isonitrogenous basis with the test protein source. Other-wise the diets are identical. After several weeks of ad libitum feeding a blood sample is taken and the results show that blood cholesterol rose with the experimental diet  The experimental techniques available The outcome of variables of interest to be assessed condition the experimental techniques to be applied, which may include growth curves, nutrient and energy balance, nutrient utilization, and signaling, etc., using cellular, molecular, or other strategies. Another approach to investigating nutritional processes is to overexpress, inactivate, or manipulate specific genes playing a role in body metabolism. These new technologies allow the study of the regulation and function of different genes. The current standard methods for manipulating genes in nutrition research depend on the method of introducing/blocking genes. Thus, genetic manipulation can be sustained for generations by creating germline transmission  Human studies In human nutrition, man is the ultimate court in which hypotheses are both generated and tested. Experimental human nutrition takes the hypothesis and through several experiments, tries to understand the nature of the link between nutrients and the metabolic basis of the disease. Once there is a reasonable body of evidence that particular nutritional conditions are related to the risk of disease, experimental nutritional epidemiology examines how population-level intervention actually influences the incidence of disease  Human nutrition experimentation The use of experimental animals for human nutrition research offers many possible solutions to experimental problems. However, the definitive experiments, where possible, should be carried out in humans. Studies involving humans are more difficult to conduct for two major reasons. First, humans vary enormously compared with laboratory animals. They vary genetically, and they also vary greatly in their lifestyle, background diet, health, physical activity, literacy, and in many other ways. Second, it is far more difficult to manipulate human diets since we do not eat purified or semi-purified diets.  Experimental diets in human nutrition intervention studies In the 1950s, an epidemiological study across seven countries presented data to suggest that the main determinant of plasma cholesterol was the balance of saturated, monounsaturated, and polyunsaturated fatty acids (MUFAs and PUFAs). To test this hypothesis, a series of studies was carried out on human volunteers using “formula diets.” Dried skimmed milk powder, the test oil, and water were blended to form a test milk with specifi c fatty acid compositions. The volunteers lived almost exclusively on these formulae. Although this type of study is simple to conduct, it does not represent the true conditions under which normal humans live. At the other end of the spectrum of options for manipulating human diets is that of issuing advice that the subjects verify by way of a food record. It is difficult to prove that subjects actually ate what they say they have eaten. Sometimes, adherence to dietary advice can be ascertained using tissue samples (blood, saliva, hair, fat) and biomarkers. For example, adherence to advice to increase oily fi sh intake can be monitored using platelet phospholipid fatty acids.  In the case of minerals and vitamins, it is possible simply to give out pills for the volunteers to take and measure compliance by counting unconsumed pills and perhaps using biomarkers. When it comes to macronutrients, this is not generally possible. Whereas asking someone to take a mineral supplement should not alter their eating habits, asking someone to consume a liter of milk a day or a bowl of rice bran per day will alter other aspects of the diets of the volunteers. It will not then be possible to attribute definitively an event to the intervention (1 l/day of milk or 1 bowl/day of rice bran). The only option in human intervention experiments is to prepare foods for volunteers to eat, which differ only in the test nutrient.  If the objective is to examine the effect of MUFAs relative to saturated fatty acids (SFAs) on blood lipids, then fat-containing foods can be prepared that are identical except for the source of the more foods and dishes that can be prepared in this way, the more successful the experiment will be. The final dilemma is where the test foods will be consumed. A volunteer may share the test foods, which are almost always supplied free of charge, with friends or family. To be sure of consumption, volunteers may be asked to consume the test meal in some supervised space, usually a metabolic suite. This, however, is a very costly option.  Study designs in human nutrition The randomized clinical trial is the most powerful design to demonstrate cause-effect relationships. It is unique in representing a completely experimental approach in humans. The major strength of randomized trials is that they are able to control most biases and confounding factors even when confounding factors cannot be measured. 23 24 25