The Evolution of Research Techniques in Premature Infant Nutrition

Buford L. Nichols, Jr.

USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, and Texas Children's Hospital, Houston, Texas 77030, USA

THE RISE OF THE TECHNOLOGY OF PREMATURE INFANT CARE

Tarnier, the French obstetrician, developed an incubator for premature infant care after seeing young chickens in an incubator at the Paris Zoo in 1878. The premature infant incubator was demonstrated in a series of technologic exhibits at World's Fairs and Expositions between 1897 and 1932. When the incubators were demonstrated for the public, premature infants were used in the display; although the drama of the presentation appealed to the general public, it appalled the medical community. Louise Recht, the nurse who managed the incubators, had been trained by Budin and used a variety of techniques to feed the infants. She demonstrated Tarnier's method of intragastric gavage and Monti's method of feeding with nasal spoons. The milk fed to the infants had usually been donated (1,2). In 1929, Pirquet, surveying the hospital management of premature infants, stated, "the main difficulty encountered in the rearing of premature children is primarily a nutritional one" (3). This chapter reviews the scientific development of premature infant feeding during an era when premature infants were placed on exhibition. I shall focus on the methods used to determine the nutritional needs of premature infants.

THE ORIGINS OF NUTRITIONAL SCIENCE

In the fifth century, Hippocrates is believed to have said, "Growing bodies have the most innate heat; they therefore require the most food, for otherwise, their bodies are wasted." This quotation from the age of antiquity sets the stage for the work of the 17th century Belgian iatrochemist Van Helmont, who used the chemical methods of his day to study problems of health. The 18th century English discoveries of carbon dioxide by Black in 1757, oxygen by Priestley in 1774, and the development of animal calorimetry by Crawford in 1779 provided the foundation for the brilliant work of Lavoisier. In 1777, Lavoisier confirmed Priestley's experimental finding that oxygen 31 32 EVOLUTION OF RESEARCH TECHNIQUES was exhausted by respiration if a sparrow were placed in a bell jar. The process was identical to that associated with the oxidation of metals. Using an ice calorimeter, Lavoisier measured the quantity of heat produced by the combustion of carbon into CO2. He continued these studies of carbon oxidation in guinea pigs and humans. After collecting gas in a closed system filled with water and inverted over a water bath, Lavoisier measured the volume of air caught and added phosphorus metal. The burning of the metal completely used up the oxygen, after which potash was introduced to remove the carbon dioxide. The remaining gas was analyzed by differences in the water level after treatment (4). The fourth century recognition of the primary elements of air, earth, fire, and water presaged the concept that air was important to life processes (4). This concept met with skepticism from Van Helmont, who combined chemistry with medicine (as had Paracelsus) and began the development of nutrition science as we know it. He coined the word "gas." In the early 18th century Stephen Hales of England included a chapter on "Analysis of Air" in his book Vegetable Statistics. Hales invented a "pedestal apparatus" bell jar to show for the first time that air became "fixed" by many organic and inorganic materials. The School of Pneumatic Chemists in England based much of its research on the work of Hales and flourished in its use of the "pedestal apparatus." In 1756, Joseph Black, one of Hales' disciples, discovered that "fixed air" or carbon dioxide was present in the mineral dolomite. Pneumatic chemistry, an area of study guided by Robert Boyle, contributed the physical laws governing the properties of gas and the discovery of "inflammable air" or hydrogen by Cavendish in 1766. Joseph Priestley described the "different kinds of air" and reported his discovery of "respirable air" or oxygen in 1772 (5). Hales' pedestal apparatus was modified by the French chemist Rouelle to allow the collection of gases. Using the technique, gases were generated in a distillation flask and the volume was estimated by the drop of the water level in the bell jar. Lavoisier used Hales' apparatus to measure the interaction of both inorganic and organic material with the volume of air to show that gases play a quantitative role in chemical reactions. He thus became the first to grasp the significance of air to chemical transformations. He used a gravimetric balance to demonstrate weight gain during the oxidation of phosphorus or sulfur and weight loss when metal oxides were reduced by high temperatures provided by a giant magnifying glass. Lavoisier observed changes in gas volume that correlated quantitatively with altered mass. His discovery of the law of conservation of mass was reported in 1771, after which air was understood to be a chemical participant in life processes (5). Lavoisier then turned his attention to the relationship between heat and oxidation. Using the bell jar and a calorimeter constructed with double layers of ice, he showed that when wood or charcoal was burned a quantitative equivalence of carbon and oxygen was consumed and carbon dioxide and heat were produced. This experiment was repeated with a living guinea pig to show that the same quantitative relationship between carbon dioxide and heat production existed in a living mammal. Using the Rouelle modification of Hales' pedestal apparatus, Lavoisier measured oxygen consumption and CO2 production in his laboratory assistant, Seguin. Lavoisier's interest EVOLUTION OF RESEARCH TECHNIQUES 33 in quantitation led to his participation in the Commission on Weights and Measures, which resulted in the centimeter-gram system of calibration units. Lavoisier contributed the definition of a calorie as the unit of heat necessary to raise the temperature of one cubic centimeter of water by one degree centigrade. Lavoisier died in 1794, a casualty of the French Revolution (4). The scientific descendants of Lavoisier continued to make elemental gas analyses using an eudiometer. Justus Liebig studied under Lavoisier's student, Gay-Lussac, from whom he learned quantitative chemistry and the endiometric techniques. He returned to his native Germany to use the new methods in animal chemistry or biochemistry studies. When Liebig undertook the proximate analysis of animal foods, tissue constituents, and isolated chemical substances using the eudiometer, new in- sights came about in rapid succession. The discovery that nitrogen was a constituent of all living matter opened the way to protein chemistry and the concept of nutrient balance. In France, Boussingault was the first to determine carbon balance in a lactating cow. Liebig carried out a nitrogen balance study in a company of the Ducal Guards in Darmstadt, Germany (4).

ENERGY NEEDS

An evaluation in France of Lavoisier's theory of calories enabled the concept of food calorie content. In 1849, Regnault and Reiset (6) extended the Hales/Rouelle bell jar experiment in a closed system with a fixed supply of air. They studied the oxygen consumption and CO2 production by a dog. Their "closed system" was used to measure the carbon oxidation of infants in a large bell jar. The Regnault-Reiset apparatus for study of oxygen utilization and CO2 production was modified in 1887 by Langlois (7) in Paris and in 1894 by Mensi in Turin (Table 1). A further evolution of this methodology was reported in France in 1908 by Weiss and in 1914 in the USA by Benedict and Talbot (9,10,17). The calorimetry energy expenditure results were less than estimates derived from studies of energy intakes in term and premature infants. In 1862 Pettenkofer and Voit in Munich developed the "open system" of indirect calorimetry. They designed a room with controlled ventilation in which an experimental subject was placed and CO2 production measured by a gravimetric procedure (18). The equipment was used by Forster (19) in 1877 to determine CO2 production in an infant. The methodology was modified and used in Berlin by Rubner and Langstein (11) in 23-hour calorimetry studies CO2 production by two premature infants at the Kaiserin Victoria Kinder Haus reported in 1915. Rubner criticized the previous work done in the closed system calorimeter because the measurements were of short duration and their emphasis was on basal metabolism rather than total carbon oxidation: "If respiratory investigations are to serve as a measure for the general consumption of nutrients, they must extend for day and night, so that the quantity of respiration corresponds to all functions of the infant" (20). The results were summarized by Levine in 1936 and 1940, who concluded that the total amount of heat 34 EVOLUTION OF RESEARCH TECHNIQUES

TABLE 1. Daily energy expenditure of premature infants

Total Author expenditure Year (location) Apparatus (calories/kg/d) Reference

1887 Langlois (Paris)8 Direct/Regnault-Reiset 81,109 7 1904 Hasselbalch Open/Pettenkofer-Vogt 31 8 (Copenhagen)3 1914 Benedict and Talbot Closed/Benedict 89-103 9,10 (Boston) 1915 Rubner and Langstein Open/Pettenkofer-Vogt 121-130 11 (Berlin) 1925 Marsh and Murlin Closed/Benedict 49 12 (Rochester) 1932 Schadow (Hamburg) Closed 45-54 13 1933 von Schlossman Closed 44-62 14 (Dusseldorf) 1936 Gordon et al. (New Closed/Benedict 58 15 York) 1940 Gordon et al. (New Open/gas analysis 68 16 York) Range 31-130

• Calculated by author from original data. produced by premature infants was lower than the amount of heat produced by term infants (Table 1) (15,16). Rubner (21) determined the laws of energy equivalency for protein, carbohydrate, and fat. His Berlin collaborator, Heubner, used the Rubner factors to determine the energy content of human milk: between 613 and 621 calories per liter (Table 2). Heubner used data on the volume of human milk consumption by term infants from studies published during the previous 20 years to calculate the energy intakes of infants. He expressed the results of these calculated intakes as an "energy quotient," with units of calories/kg per day (Table 3) (22). The energy quotient of premature infants was reported by a number of investigators between 1907 and 1920 (Table 4).

TABLE 2. Calculation of energy content of mother's milk

In 1000 g of mother's milk: 10-12 g protein = 4.1a x 10-12 = 41-49 kcal 35 g fat = 9.3a x 35 = 325.5 kcal 65 g sugar = 4.1a x 65 = 266.5 kcal Total = 633-641 kcal Round to 620 kcal/1000 g milk

From Heubner O (22). • Based upon Rubner's work. Compares with cow's milk energy content of 700 kcal/1000 g milk. EVOLUTION OF RESEARCH TECHNIQUES 35

TABLE 3. Calculation of mother's milk volume and energy quotient of term infants

Calculated energy Energy Daily milk intake quotient Author (year) volume (ml) (kcal) (kcal/kgd)

Hahner (1880) 470 + 660 291 + 409 89 + 126 Camerer and Ahlfeld (1879) 600 + 790 372 + 490 94 + 124 Camerer and Ahlfeld, (1879) 715 + 980 443 + 607 79 + 106 Ahlfeld and Hahner (1878) 660 + 850 409 + 527 95 + 123 Feer and Pfeiffer (1883,1896) 659 + 1019 408 + 632 83 + 130 641 + 1105 397 + 685 76 + 121

From Heubner 0 (22).

The values were in excess of those determined by the closed system of calorimetry, but consistent with values from open systems.

PROTEIN NEEDS

Nitrogen was discovered by Daniel Rutherford, a medical student working in 1772 at the University of Edinburgh (24). The word "protein" is derived from the Greek word proteios, meaning "first." The word was coined by Mulder to designate the nitrogen-containing substances that are constituents of all living tissues. The earliest method of proximate analysis for nitrogen consisted in collecting the gas formed in an eudiometer after oxidation with copper oxide. This method was developed by Lavoisier's disciple Gay-Lussac in 1833. In 1883, Kjeldahl published

TABLE 4. Energy intakes in premature infants

Date Author kcal per kilogram body weight/day

1907 Budin 140 average 1908 Oppenheimer 120-130 1909 Salge 130-150 1911 Samelson 115-150 1911 Oberwarth 120-160 1912 Czerny and Keller 100-120 1913 Birk 100-160 1914 Langstein and Meyer 120-130 1914 Reicke 120-130 (in those under 2000 g) 95-110 (in those over 2000 g) 1919 Moll 110-120 1920 Morse and Talbot 120 average

Overall range 95-160

From Abt IA (23). 36 EVOLUTION OF RESEARCH TECHNIQUES a method that used sulfuric acid to dissolve the organic material and leave the nitrogen in the form of ammonia. The ammonia was distilled and titrated to determine the nitrogen. A granule of zinc was added to improve distillation of the ammonia (25). This method was used in all nitrogen balance studies of premature infants and is still in use today. In 1774, Baccari isolated a protein, gluten, from wheat flour. Boussingault published the analyses of various plant proteins in 1836, after which Mulder and Liebig and his pupils continued the work. By 1842, Dumas determined that there was consid- erable variability in the elementary analyses of different proteins. His finding led to the understanding that proteins vary greatly in chemical composition and are charac- terized by a more fundamental composition from their constituent amino acids. Cyste- ine was the first of the amino acids to be discovered (Table 5) and was isolated by Wollaston in 1810 from a urinary calculus. Modern protein chemistry began, however, with the discovery of glycine as a hydrolytic product from gelatin. Kuhne determined the enzymatic hydrolysis of proteins in 1876, and Curtius found that amino acids could be linked in peptide structures of proteins. During the first 20 years of this century, Loeb demonstrated that the physical charge on an isolated protein was related to the charge of the individual amino acid constituents. His finding resulted in the understanding of the physical properties of proteins in solution. As additional information was reported about amino acid constituents within food proteins, scientists soon recognized that lysine was absent from albumins and abundant in gelatin. The acid-soluble proteins contained high percentages of glutamic acid,

TABLE 5. History of the discovery of the amino acids

Amino acid Discoverer Date Nationality

Cysteine Wollaston 1810 English Leucine Proust 1819 French Braconnot 1820 French Glycine Braconnot 1820 French Aspartic Plisson 1827 French Tyrosine Liebig 1846 German Alanine Strecker 1850 German Valine Gorup-Besanez 1856 German Serine Cramer 1865 German Glutamic Ritthausen 1866 German Phenylalanine Schulze 1879 German Arginine Schulze 1886 German Lysine Dreschsel 1889 German Histidine Kossel-Hedin 1896 German-Swedish Proline Willstatter 1900 German Tryptophane Hopkins and Cole 1901 English Hydroxyproline Fischer 1902 German Isoleucine Ehrlich 1903 German Methionine Miiller 1922 American Threonine Rose 1935 American

From McCay CM (25). EVOLUTION OF RESEARCH TECHNIQUES 37 while the proteins of hair and similar epidermal tissues have high cysteine content. Gelatin is deficient in cysteine and the aromatic amino acids. These observations led to the search for ideal protein intakes and for amino acids that cannot be synthesized in the human body (25). The first physician to collect urine from infants for nitrogen analyses was Pollak. He reported from Vienna in 1869 the collection of urine from male infants using a glass alembic (26). (An alembic is the portion of a still used to collect the condensate.) In 1875, Martin and Runge used a rubberized cloth device to collect uncontaminated urine from males, and Raudnitz used a condom for the same purpose in 1883 (27). While working under Heubner at the Charite Hospital in Berlin, Bendix (28) expanded the alembic technique to include the collection of infant stools in 1896. Although previous metabolic balance investigations had been conducted in term infants by Camerer (29) in 1878, it was not until 1910 that Langstein conducted nitrogen balance studies at the Charite in a premature infant weighing 1420 g (30). Studies were continu- ous during the first 24 days of the infant's life. Balances became positive on day 9, and weight was gained in excess of birthweight on day 17. Nitrogen balance was also included in the metabolic studies of premature infants by Rubner and Langstein (11) at the Kaiserin Victoria Kinder Haus in Berlin in 1915 (Table 6). The next investigation of nitrogen balance in premature infants was reported from Schlossman's Clinic in Diisseldorf in 1931. The first infant weighed 1930 g and was studied from the 23rd day of life. During the next 9 days, daily protein retention was 3.2 g/kg while weight increased from 2420 to 2660 g. The second infant, born at 1170 g, was studied from the 5th to the 14th day after birth. Average daily retention of protein was 1.6 g/kg. The infant's weight increased from 1480 to 1660 g during the balance study (32). Following this pioneer work, many others reported on nitrogen balance: Gulacsy (33) in 1932, Tanz and Unger (34) in 1937, and Levine in the United States in 1937 (35) (Table 7). Gordon (35) studied six healthy premature infants between 16 and 53 days of age. Daily retentions of approximately 1.5 g protein per kg were observed in four infants who received human milk. Similar retentions of cow's milk protein were observed when diets contained similar energy and protein contents.

TABLE 6. Early nitrogen balances in term infants

Author Location Date Age Diet

Lange Leipzig 1895 1-8 mo Artificial milk3 Michel Paris 1896 4-14 d Mother's milk Bendix Berlin 1896 3 mo Artificial milk" Lange Leipzig 1897 6-7 mo Artificial milk8 Rubner and Heubner Berlin 1897 2 mo Mother's milk3 Groz Budapest 1897 5-15 d Artificial milk* Keller Breslau 1898 5-8 mo Artificial milk3 Rubner and Heubner Berlin 1899 Artificial milk*"

From Keller A (31). • Kjeldahl method. 38 EVOLUTION OF RESEARCH TECHNIQUES

TABLE 7. Early nitrogen balances in preterm infants

Author Location Date Reference

Langstein and Niemann Berlin 1910 30 Rubner and Langstein Berlin 1915 11 von Schlossman Dusseldorf 1931 14 Gulacsy Budapest 1932 33 Gordon, Levine, et al. New York 1937 35 Tanz and Unger Vienna 1938 34

As indicated in Table 4, the discovery of the amino acids peaked at the beginning of the 20th century. In 1917, researchers realized that the composition of milk proteins differed in at least four essential amino acids (Table 8). In Berlin, Edelstein and Langstein (36) conducted feeding studies in five healthy term infants from 2 to 4 months of age to determine the biological value of individual human and cow's milk proteins and casein and lactalbumin from cow's milk. The investigators found that 88% of dietary nitrogen was retained from mother's milk, but only 73% from whole cow's milk. The results for lactalbumin and casein were 87% and 73%, respectively. The nitrogen balance values were paralleled by weight gain in the experimental sub- jects. The significance of this investigation was summarized in the title of the paper, "The protein problem." Langstein concluded that human milk was a better substrate than cow's milk for infant growth. Similar studies in the USA by Gordon, Levine, and co-workers (35) in 1937 failed to confirm Langstein's findings when dietary energy and protein intake were held constant. In the six premature infants studied by Gordon, Levine, et ah, retention of human milk was 70.9% and cow's milk was 70.5%. Further studies on amino acid requirements of premature infants were not conducted until the 1960s (37).

TABLE 8. Earliest experiments on milk protein quality in infants

Cow's milk protein composition Amino acid Casein Lactoglobulin Lactalbumin

Cysteine-A/ 1.30% 1.90% 2.18% Lysine-A/ 9.46% 8.58% 12.54% Arginine-/V 9.31% 10.70% 7.56% Histidine-A/ 6.55% 3.96% 4.44%

Nitrogen retentions (% of intake) Lactalbumin Casein Mother's milk Cow's milk

94.91 73.55 88.4 73.7 88.3 93.69 78.00 72.27 81.84 73.0 Mean 87 73 88 73

From Edelstein F and Langstein L (36). EVOLUTION OF RESEARCH TECHNIQUES 39

In 1946, Levine (38) reported that premature infants excreted increased quantities of an unknown substance in the urine. This material gave a false-positive test for creatinine and was identified as p-hydroxyphenyllactic and p-hydroxyphenylpyruvic acids. The quantity excreted was directly linked to a daily cow's milk protein intake of more than 5 g per kg. Excretion of the acids disappeared when the high protein diet was supplemented with 10 to 20 mg of ascorbic acid daily. These experiments revealed that amino acid catabolism and tyrosine synthesis was immature in premature infants (38).

ACKNOWLEDGMENTS

I thank Professor Hans Helge of the Free University and Director of the Kaiserin Victoria Kinder Haus, Berlin, for his invaluable and enthusiastic support in research- ing the development of pediatrics in Berlin, Mrs. Bee I. Wong of the Children's Nutrition Research Center (CNRC) library for her diligence in obtaining the older references, and E. R. Klein of the CNRC editorial office for editorial assistance. This work is a publication of the USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas, USA. Funding has been provided from the USDA/ARS under Co- operative Agreement No. 58-6250-1-003. The contents of this publication do not nec- essarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

REFERENCES

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DISCUSSION

Dr. Fukagawa: I should like to ask about the actual energy requirements of the premature infant. The range you gave was between 40 and 100 to 120 kcal/kg.d. In practice, many neonatologists wrestle with the problem of whether or not it is practical to try to achieve daily intakes of over 100 kcal/kg. If we use supplementary parenteral nutrition to try to achieve these intakes we often run into problems of fat babies or fatty livers. Dr. Nichols: As you know there is no strongly accepted concept of what constitutes an adequate intake of any nutrient. The first question to ask is, What is the usual intake? The usual intake fails in the newborn period and during fasting, and it certainly failed during the period of starvation of the premature infant that used to be traditional before the 1960s. But how much is enough? We continue to debate this. One view is that you should always feed EVOLUTION OF RESEARCH TECHNIQUES 41 enough to maintain balance, which is of course always positive in growing babies. Another is that you should aim to meet the intrauterine growth rate. If we had a functional outcome, such as improved immunological function, then we could have a more objective basis on which to establish adequacy of intake. Dr. Jequier: Modern research has shown that the limits of energy expenditure are much tighter than indicated by those broad limits of 40 to 120 kcal/kg.d, when you take into account body weight and composition and the age of the infant. Using modern techniques we can now predict quite accurately what the energy requirements are. Dr. Nichols: Maybe I didn't make this point sufficiently clear. Rubner pointed out that if you are studying metabolism, basal metabolic rate may have some functional meaning, but if you want to know energy expenditure over a period of time you have to use some other system. This is the major difference between the reports I cited—whether you measure the basal metabolic rate and extrapolate that to a 24-hour energy expenditure, or whether you measure expenditure over a longer period as Rubner did. There are many more papers on basal metabolic rate than there are on total energy expenditure. Your point is well taken that we can now measure energy expenditure over longer periods and with greater precision than was previously possible. We now come up with energy expenditure values that are much closer to the energy intakes of premature infants. Dr. Orzalesi: How did the early investigators attempt to relate their results to aspects such as the size of the baby, the age of the baby, and the environment in which the baby was nursed? Dr. Nichols: Talbot carried out the most extensive series of investigations into the effects of gestational age and body weight. His studies were strictly based on basal metabolic rate (BMR), which he tried to extrapolate to 24 hours. He divided activity into four categor- ies—crying, restlessness, resting, and postprandial—and would then try to extrapolate these to the whole day. Unfortunately the results were much lower than the actual expenditure during that period. However, he did feel that there was an increase in energy expenditure in low birthweight infants during the early postnatal period. One particular child whom he studied, a baby weighing 1800 g, showed a gradual rise in metabolic rate over the study period, which correlated with studies of nitrogen balance carried out in the same subject. As to the environment, none of the early studies was very precise. It was only when Gordon and Levine published their work in 1941 that information became available on the effects of systematic variation in environmental temperature on carbon dioxide production. Dr. Boyd: We live in an age when we tend to think we are at the end of history. We review all our own studies in a very narrow frame and it is unusual to give as much time and effort to the historical basis of our knowledge as you have done. Has the design of your own investigations been much influenced by your historical knowledge? Dr. Nichols: As you go back into these issues you find the truth being reinvented and the work of the pre-First World War German investigators being completely ignored. Yet the quality of their work was quite acceptable even by today's standards. I think there is much we can learn from the past. Dr. Priolisi: Work done by Hevessy here in Palermo in 1938 should be recognized. In that year he published in the Journal of Biochemistry the first paper on the use of radioisotopes to investigate metabolism (a study of phosphorus metabolism in animals). He subsequently emigrated to the United States. Dr. Nichols: Dr. Hevessy was also the person who introduced the K40 method for measur- ing lean body mass. He was a remarkable scientist.