Introduction

Growth hormone (GH) is secreted by the anterior pituitary in a pulsatile fashion. Its secretion is tightly regulated by hypothalamic factors and by feedback from peripheral factors such as serum glucose and fatty acid levels. The hypothalamic input includes the reciprocal secretion of somatostatin and growth hormone releasing hormone (GHRH). Thus, a pulse of GH is mediated by suppression of tonic hypothalamic somatostatin secretion associated with an increase in GHRH secretion. This will not be further discussed but has been reviewed in detail previously (Tannenbaum & Ling, 1984; Thorner et al., 1986). As shown in Figure 1, GH is also regulated by circulating levels of somatomedin C (otherwise known as insulin-like growth factor) which is either produced locally or in the periphery. Thus, somatomedin C inhibits GH secretion at both the pituitary and hypothalamic levels by modulating somatostatin and possibly also GHRH secretion. Another important influence on GH secretion is gonadal steroids. This occurs in both the human and in animals.

Growth hormone secretion during the life cycle in the human

Levels of GH are detectable in the fetus during the mid-trimester and remain high throughout intrauterine life. Detectable GH is found in the serum of human fetuses as early as 70 days of gestation and by mid to late second trimester, values may reach 150 ng/ml. Thereafter, GH levels decline, but at the time of birth and for several weeks thereafter, the levels remain high when compared to adult values. Following delivery, GH levels fall and remain relatively low during childhood and rise again at the time of puberty.

Introduction

The somatomedins or insulin-like growth factors are a family of growth-promoting peptide hormones consisting of insulin-like growth factors I (IGF-I) and II (IGF-II) as well as their variant forms (Hall & Sara, 1983). The first members of this family to be characterized were isolated from human adult plasma by Rinderknecht and Humbel (1978a,b) and termed IGF-I and IGF-II due to their structural homology to insulin. IGF-I and IGF-II are homologous single chain peptides with intrachain disulphide bridges and consist of 70 and 67 amino acids respectively. Several variant forms of these peptides have been identified at both the cDNA and protein level (Jansen et al, 1985; Zumstein, Luthi & Humbel, 1985).

The somatomedins were first suggested to play a role in neural function prior to their characterization. Studies of hormonal influences on brain development led to the hypothesis that brain growth was regulated by a growth factor whose production could be stimulated by growth hormone (Sara & Lazarus, 1974; Sara et al, 1974). In a tempts to characterize this brain growth factor it became obvious that it was a member of the somatomedin family (Sara et al., 1976). It took many years however until this factor could be isolated and its amino acid sequence established (Sara et al, 1986). During this time it also became clear that the factor was not only involved in the regulation of brain growth but also was involved in the maintenance of the mature nervous system (Sara, Hall & Wetterberg, 1981).

In this review, the role of the somatomedins as neuropeptides will be discussed, with emphasis being placed upon their characterization and biosynthesis as well as their biological role in the nervous system.

Standardisation of basic measurements

Many instruments for basic physical measurements can be purchased with accuracy certified by the National Physical Laboratory in the UK, the National Bureau of Standards in the USA, or from equivalent authorities in some other countries. Instruments such as mercury-in-glass thermometers, mercury barometers, certified weights and resistance boxes should maintain their accuracy indefinitely; they may be used for regular calibrations of thermistors, thermocouple- or resistance-thermometers, manometers or pressure sensors, electronic balances, and resistors. Other electronic instruments require to be checked against a reliable standard at least once a year and often more frequently; this can be done by some suppliers who are approved by their national standards authority. A high-quality, multirange digital voltmeter, regularly serviced by an approved supplier, is now an essential piece of laboratory equipment for calorimetry, and has virtually replaced the old standard cell and potentiometer. It may be used as a secondary voltage standard for the calibration of potentiometric recorders as well as of a great many instruments which provide output in voltage analogue form. For direct calorimetry a high quality wattmeter is equally essential.

Calibration of gas analysers

Introduction

Accurate calibration of gas analysers provides one of the greatest challenges in calorimetry; it is an area whose importance is not always fully appreciated even amongst some workers engaged in calorimetry. The calibration methods used and the care with which calibration is carried out, are often inadequate to achieve the appropriate level of accuracy for their experiments.

Calorimetry is the measurement of heat. By means of animal calorimetry (the term ‘animal’ here includes humans) we can estimate the energy costs of living. All life processes including growth, work and agricultural production (milk, eggs, wool, etc.) use energy, the source of the energy being food. The energy content of food is metabolised (i.e. changed) in the body into other energy forms, only some of which are useful in the sense of growth or production. Much of the wasted energy is given off from the body in the form of heat – hence the name calorimetry.

The heat may be measured directly by physical methods (Direct Calorimetry) or it may be inferred from quantitative measurement of some of the chemical by-products of metabolism (Indirect Calorimetry). These alternatives are possible because of the natural constraints imposed on energy transformations by the laws of thermodynamics. Of fundamental importance are the Law of Conservation of Energy (energy cannot be created or destroyed, only changed in form) and the Hess Law of Constant Heat Summation (the heat released by a chain of reactions is independent of the chemical pathways, and dependent only on the end-products). In effect these laws ensure that the heat evolved in the enormously complex cycle of biochemical reactions that occur in the body is exactly the same as that which is measured when the same food is converted into the same end-products by simple combustion on a laboratory bench or in a calorimeter.

The partition of the gross energy of food into its major energy sub-divisions is illustrated in Fig. 1.1. Some food is undigested resulting in a loss of energy as faeces.

At the first Symposium on Energy Metabolism a small committee (K. L. Blaxter, Scotland; K. Nehring, Germany (DDR); W. Wöhlbier, Germany (DBR); E. Brouwer, Netherlands) was appointed to consider and to recommend constants and factors to be used in calculations of energy metabolism. After studying the problem a provisional set of constants and factors was sent for criticism to the participants of the symposium. No objections were raised. The committee therefore considers its main task to be finished and makes the following recommendations.

Direct animal calorimeters measure the total heat generated inside them and partition the heat into its two components, evaporative and non-evaporative. Non-evaporative or sensible heat is heat given off from an animal by radiation to surrounding surfaces, by convection to the surrounding air and by conduction to any objects with which the animal is in contact. Evaporative heat loss occurs because the conversion of liquid water into vapour requires heat energy. The latent heat of vaporisation is the heat required to vaporise unit mass of water. When water is vaporised in the respiratory passages during normal respiration and panting, or at the skin surface during perspiration, the latent heat of vaporisation is derived mainly from the animal. This heat loss by the animal is transferred to the air in the form of increased humidity; the enthalpy of the air (which is a measure of its energy content and depends on temperature, humidity and pressure) is increased.

The psychrometric chart

The relationships between temperature, humidity and enthalpy of air are often illustrated in the form of a psychrometric chart. Fig. 5.1 is a scaled down version of a psychrometric chart for standard barometric pressure. The heavy line is a plot against temperature of the mixing ratio (i.e. humidity expressed as weight of water per weight of dry air) for air saturated with water vapour; it represents the maximum amount of vaporised moisture that air may contain.

Measurement of the rate of metabolic heat production by indirect calorimetry depends on two assumptions:

1. (1) It is assumed that the end result of all the biochemical reactions which occur in the body amounts effectively to the combustion or synthesis of three substances – carbohydrate, fat and protein.

2. (2) It is assumed that for each of these substances, when it is oxidised in the body, there are fixed ratios between the quantities of oxygen consumed, carbon dioxide produced and heat produced.

By any standards these are gross oversimplifications. The first completely ignores the metabolism of minerals, which represent nearly 7% of the total bodyweight. Although the mineral status of the body may be nearly stable in unproductive adults, major changes occur during phases of skeletal growth, pregnancy and lactation. The second assumption implies a uniformity in the properties of fat and protein that would seem improbable in view of their extremely varied chemical compositions. The only real justification for the assumptions is that over many years of practical application it has been found that indirect calorimetry is remarkably consistent and in close agreement with direct calorimetry.

The first step in evaluating a theoretical basis for indirect calorimetry must be to establish the values of the calorific factors for carbohydrate, fat and protein. These are determined from the results of combustion of the materials in a bomb calorimeter.

Bomb calorimetry

Adiabatic bomb calorimetry

As the name of the calorimeter implies, the energy content of a sample is determined by combustion in a closed system which can neither gain heat from, nor lose heat to the outside environment.

Whilst direct calorimeters measure the rate of heat dissipation of a subject, indirect calorimeters measure the rate of heat generation; averaged over a long period of time the two rates will be equal or very nearly so. Thus it is a mistake to believe that because of its name, indirect calorimetry is a second-rate means of measuring heat production.

Indirect calorimetry estimates heat production from quantitative measurements of materials consumed and produced during metabolism. Most methods involve estimation of respiratory gas exchange and these may be classified according to their operating principles as confinement, closed-circuit, total collection, and open-circuit systems.

In confinement systems the subject is held in a sealed chamber and the rates of change of gas concentration in the chamber are recorded.

In closed-circuit systems the subject is again held within or breathes into a sealed apparatus; the carbon dioxide and water vapour produced by the subject are measured as the weight gain of appropriate absorbers, and the amount of oxygen consumed is measured by metering the amount required to replenish the system. In total collection systems all the air expired by the subject is accumulated in order to measure subsequently its volume and chemical composition.

There are two major forms of open-circuit calorimetry. In one the subject breathes directly from atmosphere and by means of a non-return valve system expires into a separate outlet line. In the second form, the subject inspires from, and expires to, a stream of air passing, by means of a pump or fan, across the face.

Closed-circuit or confinement chambers for measuring oxygen consumption of small animals can be constructed mainly from standard laboratory ware. The system described by Smothers (1966) is a good example. Alternatively some degree of automation may be added (e.g. Heusner et al., 1971; Stock, 1975). The classical gravimetric method of Haldane (1892) combined with a modern electronic balance has still much to commend it. All of these chambers may be easily assembled without resort to expensive equipment, but the animal is confined and cannot readily be handled; and measurements are restricted to collection periods of short duration. Whilst these methods are valuable in some circumstances, they should be used with an awareness of their limitations and the results obtained with them interpreted with caution. For example, our experience is that scaling up a few short measurement periods does not give an accurate estimation of 24 h energy expenditure; and in addition the animals may take some time to adapt to their new housing and measurements over this period may be misleading.

For fast response measurements, particularly on humans and large animals, and for long-term studies, an open-circuit system employing flowmeters, electronic gas analysers and recording equipment is necessary. Various combinations of equipment are possible depending on the degree of accuracy required and the finance available. In this chapter we describe a few complete systems that have been found to operate satisfactorily, and which could be adopted in part or in entirety by the reader.

The impetus for writing this book was born out of a realisation that expertise in calorimetric techniques relied for its distribution largely on chance conversations between a limited ‘club’ of practitioners. Many research workers in medicine and other fields of biology have embarked on calorimetry in the mistaken belief that measurement of oxygen consumption and hence energy expenditure is a very straightforward process. It is almost certain that results and conclusions which have been reported from some calorimetric systems are incorrect. There has been no comprehensive text-book, and newcomers to the field have usually acquired knowledge by visiting establishments where calorimeters already exist. Sub-sequently, they have based their own equipment on what they have seen, sometimes unaware that a quite different form of calorimeter might be better suited to their particular needs. Furthermore, practitioners in human calorimetry and farm animal calorimetry seldom meet and have tended to develop their ideas along different lines. Complete systems for calorimetry were, and indeed still are, rarely available commercially and some commercial instruments are of poor accuracy; some indeed are based on incorrect scientific principles. Whether buying a complete system or building one up from components, it is necessary to have a full understanding of the basic principles and of the many sources of error that must be guarded against. We hope that this book will be useful to teachers and students as well as those embarking on calorimetry. The extensive treatment that we have given to the measurement of gas concentrations and flow-rates should also be useful to respiratory physiologists.