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( .- (1'1“ "‘11, MI, ‘ 3 5.; 7;? im‘ 1’ _ I i I i’ 23-) .. _ Zr 1 ul ; ’ - i E 4?"; .5; G t. R' ?‘ . . . . .. 1’” r' l ' «(I ‘ " I’.l d . _‘ ‘:‘I. .9 g (7; '~ A ~ 0. ? '.\ 52" ‘3 , m‘ 'g. ‘41..lv-.‘.‘ . . . ,;- a .: .t- ' . '. .n'. at '3» .4 J‘ ‘ :S h ‘4? ' .44“ L "1:" 3‘ ’34! 1;;“35 i l.- L‘w a f E u‘ E '4'. 9'. mafia g; . :2 ,M. It; 9‘- H z): _ 33V}; 4 \49 .5. t; ,3 y \g; 2:: 5;." .. a}; 6" z mT-Efim'i“ K'fl.m-ITM"€JW€I Wugvflryty If 1 v . This is to certify that the dissertation entitled a The Influence of Zinc on Growth and Development and on Energy Intakes of Children with Chronic Renal Failure presented by Dorothy wermuth Hagan has been accepted towards fulfillment of the requirements for Eh I). degreein Human Nutrition @M: Adm mg /, Major professor Date //"/.S.' (Pg MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES -— v RETURNING MATERIALS: Place in book drop to remove this checkout from your record. ‘FINES will be charged if book is returned after the date stamped below. r, THE INFLUENCE OF ZINC ON GROWTH AND DEVELOPMENT AND ON ENERGY INTAKES OF CHILDREN WITH CHRONIC RENAL FAILURE BY Dorothy Wermuth Hagan A DISSERTATION Submitted to Michigan State University in paIWLial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1985 ABSTRACT THE INFLUENCE OF ZINC ON GROWTH AND DEVELOPMENT AND ON ENERGY INTAKES OF CHILDREN WITH CHRONIC RENAL FAILURE BY Dorothy Wermuth Hagan This investigation assessed whether zinc acetate supplementation (2mg/kg BW, maximum 40 mg/da/child) in Children with End Stage Renal Disease, improved energy intakes and, in turn, growth and development. Twelve children completed the study. Three of the twelve did not receive zinc supplement. Seven of the 9 supplemented children, were followed for 1 year before supplementation and 1 year after supplementation. The remaining 2 subjects were followed for shorter periods of time both pre- and post—supplementation. Height, weight, mid-arm circumference, triceps fatfold, hand wrist radiographs, and Tanner Staging measurements, were taken at the beginning of the study, prior to zinc supplementation, and at the end of the study period. Clinical analyses for serum sodium, chloride, potassium, calcium, phosphorus, magnesium, alkaline phosphatase, total protein, albumin, blood urea nitrogen, creatinine, and 002 were routinely completed monthly. Simultaneously, plasma zinc and copper and erythrocyte zinc and 3 day food diaries were conupleted. Food diaries were then analysed for energy, protein, calcium, iron, phosphorus, sodium, potassium, and zinc ithakes using the Michigan State University Nutrient Data Bank and compared to the standards resulting from HANES I and II data. Mean growth velocity in males was 4.07 +/— 2.02 cm/yr (non-supplemented), 2.98 +/— 2.33 cm/yr (supplemented) and in females, 3.88 +/- 0.73 cm/yr (non—supplemented), 3.28 +/- 2.10 cm/yr (supplemented). There were no significant differences between the supplemented and non—zinc supplemented males or females in growth velocity. Bone maturation as determined through hand wrist radiographs, improved in 4 of 6 zinc supplemented subjects. Mean plasma zinc and copper levels before zinc supplementation were 97.1 +/- 17.1 mcg/dl and 164.2 mcg/dl, and after supplementation, 101.9 mcg/dl and 172.8 mcg/dl, respectivley. There were no significant differences in the plasma zinc or copper levels with or without zinc supplementation. Before zinc supplementation, 50%, 92% and 42 % of the subjects met 67% of their RDA for age and sex for energy, protein, and zinc, repectively. After zinc acetate supplementation, the percentage of subjects meeting 67% of the RDA for energy, protein, and dietary zinc were 67%, 100%, and 67%, respectively. There was a trend toward increased dietary energy, protein, supplementation. and zinc intake with zinc acetate DEDICATION This dissertation is dedicated to twenty—six children with Chronic Renal Failure at Children's Hospital of Michigan who participated in this investigation. In working with these children over more than a two year period, one is impressed with their bravery and maturity. Although like other children in most ways, they had to accept many disappointments and setbacks, additional responsibility for their future, and physical and psychological pain. Their desire to be "normal" is high, especially in males as it relates to attainment of an acceptable adult height. These children were often inconvenienced and made many sacrifices by participating in this investigation. Their contribution to increased knowledge will benefit future children with Chronic Renal Failure. This investigator and future benefactors appreciate their help and sacrifice. ii ACKNOWLEDGEMENTS The successful completion of this project took the cooperation of four major institutions in the state of Michigan: Michigan State University, Children‘s Hospital of Michigan, Wayne State University School of Medicine, and the Veterans Administration Hospital, Allen Park. Michigan State University provided excellent support for the successful completion of this project. Dr. Jenny Bond, Dr. Wanda Chenoweth, and Dr. Rachel Schemmel each understood the problems associated with a human study and provided encouragement and suggestions as problems arose. Dr. John Vinsonhaler was instumental in teaching computer literacy and systems analysis which were used in the selection and operation of computer equipment used for manuscript preparation. The Administration of Children's Hospital of Michigan, particularly, Dr. Robert Gregg, former president, and Mr. Paul Broughton, current president, approved and supported the conducting of the research project at the hospital. Dr. Larry Fleischmann participated in guidance committee activities, and assisted in patient participation, data iii collection, and clinical instruction and advice. Not only is he a brillant physician and an excellent teacher, but he was also an advocate for utilizing allied health professionals in the care of renal patients long before it became popular and has built an effective team to provide ongoing care. This team was most helpful in collecting and coordinating data needed for this project. Special mention must be given to Nancy Gentner McDonald, Clinical Dietitian, Debbie McWilliams, Jan Sfeftick, and Mary Ann Lynch, Nursing. Information on methods and procedures used in routine laboratory analyses was provided by Mr. Abner Robinson, former, Director of Chemistry and Associate Diretor of Laboratories, and Dr. William Ferrel, current director. The pharmacy staff, particularly Gary Mark, consulted on the preparation of the zinc supplement. Dr. Ananda Prasad from Wayne State University and the Veterans Administration Hospital, consulted on the research design, laboratory analysis of zinc and copper, and zinc supplementation. His laboratory staff at both the Veterans Administration Hospital and Wayne State University were most helpful in teaching the laboratory procedures for zinc and copper analysis in plasma and erythrocytes. Pat Flouriany was particularly helpful in the processing of blood samples OVer a time period in excess of two years. iv This project was supported in part by Dietitians in Pediatric Practice, The American Dietetic Association. Dr. Larry Fleischmann supported the costs involved with zinc and copper analyses. Dr. Rachel Schemmel, Michigan State University supported computer costs through an agricultural research grant. Dr. Rachel Schemmel, my major professor, provided excellent guidance throughout the doctoral program. Her constant encouragement through each phase of the education process was instumental in the successful completion of the academic and research requirements. I am truely appreciative. Last, but not least, my husband, Bill, has foregone many social functions and vacations and accepted additional home responsibilities so that my personal goal could be achieved. Again, I am most grateful. LIST OF LIST OF I. II. III. IV. TABLE OF CONTENTS TABLES .......................................... FIGURES ......................................... INTRODUCTION .................................... LITERATURE REVIEW A. Growth ...................................... 1. Introduction ............................ 2. Standards for Measuring Growth .......... B. Growth and Chronic Renal Failure ............ 1. Introduction ............................ 2. Growth Implications ..................... C. Growth, Renal Disease and Zinc .............. 1. Introduction ............................ 2. Growth and Zinc ......................... 3. Zinc and Renal Disease .................. D. Analysis of Zinc ............................ METHODS AND PROCEDURES A. Research Site ............................... B. Subjects .................................... C. Dialysis Fluids ............................. D. Anthropometric Measurements ................. E. Radiographs ................................. F. Physical Examination ........................ G. Laboratory Analysis ......................... H. Zinc and Copper Analysis .................... I. Food Intakes ................................ J. Zinc Supplement ............................. K. Statistical Procedures ...................... RESULTS A. Introduction ................................ B. Growth Measurements ......................... C. Anthropometric Measurements ................. D. Chronological Age and Bone Age Comparisons.. E. Tanner Staging .............................. F. Biochemical Measurements .................... V1 Page 71 105 VI. VII. VIII. IX. G. Dietary Measurements ........................ 65 H. Growth Evaluation ........................... 68 DISCUSSION OF RESULTS A. Introduction ................................ 107 B. Growth Measurements ......................... 109 C. Biochemical Measurements .................... 113 D Dietary Measurements ........................ 122 SUMMARY AND CONCLUSIONS ......................... 126 SUGGESTIONS FOR FURTHER RESEARCH ................ 128 BIBLIOGRAPHY .................................... 130 APPENDIX A ...................................... 144 APPENDIX B ...................................... 149 vii LIST OF TABLES Table Page 1. Normal Ranges of Blood Chemistries for Children at Children's Hospital of Michigan ............... 71 2. Common Medications Used in Treatment of ESRD at Children's Hospital of Michigan ............... 72 3. Characteristics of Study Population .............. 73 4. Composition of Dialysate Solutions Used at Children's Hospital of Michigan .................. 74 5. Disease Characteristics of Participants .......... 75 6. Ages, Heights and Weights of Subjects at Initiation of Study .............................. 76 7. Prediction of Adult Height By Various Methods for Male and Female Subjects ......................... 77 8. Growth Velocity — Males .......................... 78 9. Growth Velocity - Females ........................ 79 10. Height per Age, Weight per Age of Male and Female Subjects expressed as Z—scores ............ 80 11. Correlation Coefficients for Height per Age, Weight per Age, and Weight per Height for Male and Female Subjects .............................. 81 12. Weight per Height of Male and Female Subjects Expressed as Percent Median ...................... 82 13. Arm Muscle Circumference (AMC) Expressed as Percent Median, Mid—Arm Circumference (MAC), and Triceps Fatfold (TSF) Expressed as Z—scores for Male and Female Subjects ......................... 83 14. Chronological Age (CA) and Bone Age (BA) for Males ............................................ 84 15. Chronological Age (CA) and Bone Age (BA) for viii 16. 18. 19. 20. 21. 22. 23. 25. 26. 27. 28. 29. 30 Females .......................................... Comparison of Slope for Growth Velocity (CV) and Bone Matuation (BM) for Male and Female Subjects ......................................... Sexual Maturity Ratings for Males Expressed as Tanner Score and Centile ......................... Sexual Maturity Ratings for Females Expressed as Tanner Score and Centile ......................... Individual Means for BUN and Serum Creatinine for Male and Female Subjects ..................... Individual Means for CO and Alkaline Phosphatase (Alk Phos) or Male and Female Subjects ......................................... Comparison of Alkaline Phosphatase (A/P) and Plasma Zinc Levels for Male and Female Subjects.. Individual Means for Serum Sodium and Serum Chloride for Male and Female Subjects ............ Individual Means for Serum Calcium and Serum Phosphorus for Male and Female Subjects .......... Individual Means for Serum Potassium and Serum Magnesium for Male and Female Subjects ........... Individual Means for Hemoglobin and Hematocrit for Male and Female Subjects ..................... Individual Means for Total Serum Protein and Serum Albumin for Male and Female Subjects ....... Individual Mean Plasma and RBC Zinc and Plasma Copper Levels for Males .......................... Individual Mean Plasma and RBC Zinc and Plasma Copper Levels for Females ........................ Group Mean Zinc and Copper Blood Levels for Male and Female and Males and Females Combined ........ Individual Mean Dietary Intakes for Males Evaluated as Percent Median of HANES II .......... 1x 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 31. 32. 33. 34. Individual Mean Dietary Intakes for Females Evaluated as Percent Median of HANES II .......... Dietary Intakes for Kilocalories (Kcal), Protein (Pro), Calcium (Ca), Iron (Fe) for Male and Female Subjects Expressed as Z-scores (2).... Recommended Nutrient Levels and Percentage of Recommended for Male and Female Subjects ......... Relationship of Z-scores for Height, Age of Onset of ESRD, and Duration of Dialysis for Males and Females .......................................... 101 103 104 LIST OF FIGURES Figures Page 1. Growth Velocity of Subjects With and Without Zinc Acetate Supplementation ..................... 105 2. Mean Skeletal Age at Each Chronological Age (Diagonal Line) From Greulich and Pyle. Radiographic Atlas of Skeletal Development of the Hand and Wrist, 1959. Subjects Before ZN - *. Crossover Point — 0, After ZN — [] ............... 106 3. Hand Wrist Radiograph of Subject 5 Before Zinc Supplementation: Chronological Age 9.7 Years; Bone Age 3.5 Years .............................. 150 4. Hand Wrist Radiograph of Subject 5 After Zinc Supplementation: Chronological Age 9.7 Years; Bone Age 4.5 Years ............................... 151 5. Hand Wrist Radiograph of Subject 10 Before Zinc Supplementation: Chronological Age 10.7 Years; Bone Age 8.5 Years ............................... 153 6. Hand Wrist Radiograph of Subject 10 After Zinc Supplementation: Chronological Age 11.4 Years; Bone Age 9.0 Years ............................... 154 7. Hand Wrist Radiograph of Subject 17 Before Zinc Supplementation: Chronological Age 10.4 Years; Bone Age 5.0 Years ............................... 156 8. Hand Wrist Radiograph of Subject 17 After Zinc Supplementation: Chronological Age 11.3 Years; Bone Age 6.0 Years ............................... 157 9. Hand Wrist Radiograph of Subject 15 Before Zinc Supplementation: Chronological Age 13.2 Years; Bone Age 9.5 Years ............................... 159 10. Hand Wrist Radiograph of Subject 15 After Zinc Supplementation: Chronological Age 14.0 Years; Bone Age 11.0 Years ............................... 160 xi 11. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Hand Wrist Radiograph of Subject 1 at Beginning of Study Period: Chronological Age 4.2 Years; Bone Age 4.0 Years ................................ Hand Wrist Radiograph of Subject 1 at End of Study Period: Chronological Age 5.5 Years; Bone Age 4.5 Years ................................ Hand Wrist Radiograph of Subject 9 at Beginning of Study Period: Chronological Age 13.8 Years; Bone Age 8.5 Years ................................ Hand Wrist Radiograph of Subject 9 at End of Study Period: Chronological Age 14.7 Years; Bone Age 10.0 Years ............................... Hand Wrist Radiograph of Subject 14 at Beginning of Study Period: Chronological Age 15.8 Years; Bone Age 11.5 Years ............................... Hand Wrist Radiograph of Subject 14 at End of Study Period: Chronological Age 16.5 Years; Bone Age 12.0 Years ............................... Hand Wrist Radiograph of Subject 18 Before Zinc Supplementation: Chronological Age 14.9 Years; Bone Age 13.5 Years ............................... Hand Wrist Radiograph of Subject 18 After Zinc Supplementation: Chronological Age 15.6 Years; Bone Age 15.0 Years ............................... Hand Wrist Radiograph of Subject 19 Before Zinc Supplementation: Chronological Age 15.5 Years; Bone Age 9.0 Years ................................ Hand Wrist Radiograph of Subject 19 After Zinc Supplementation: Chronological Age 16.4 Years; Bone Age 11.0 Years ............................... Hand Wrist Radiograph of Subject 23 Before Zinc Supplementation: Chronological Age 6.4 Years; Bone Age 4.2 Years ................................ Hand Wrist Radiograph of Subject 24 Before Zinc Supplementation: Chronological Age 12.8 Years; Bone Age 10.0 Years ............................... xii 162 163 165 166 168 169 171 172 174 175 177 179 23. 24. Hand Wrist Radiograph of Subject 24 After Zinc Supplementation: Chronological Age 13.7 Years; Bone Age 10.0 Years ............................... Hand Wrist Radiograph of Subject 26 Before Zinc Supplementation: Chronological Age 20.9 Years; Bone Age 17.0 Years ............................... xiii 180 182 I. INTRODUCTION Chronic Renal Failure (CRF) has been associated with retarded growth in children (West and Smith, 1956; Betts and Magrath, 1974; Mehls et al, 1978; Potter and Greifer, 1978). In comparing a number of studies, Potter and Greifer (1978), report growth failure in 35 to 65 percent of all children with CRF. Growth failure in children with CRF has been associated with azotemia, acidosis, hyposthenuria, renal osteodystrophy, endocrine dysfunction, energy deficiency, abnormalities of protein metabolism, and treatment by glucocorticoid therapy (Potter and Greifer, 1978). Reports in the literature relative to the effect of energy levels are controversial. Betts and Magrath (1974) found a significant correlation (r=0.72; p<0.001) between growth velocity, expressed as percentage of expected 50th percentile, and energy intake, expressed as percentage of kilocalories recommended for age. Normal growth would be expected in their study with an energy intake above 80 percent of kilocalories recommended for children in the United Kingdom. Likewise Simmons et al. (1971) determined that 70 percent of the Recommended Dietary Allowances (RDA) for energy must be consumed by uremic children to achieve growth. Subsequent work by Betts et al. (1977) showed that a mean energy increase of 8.4 percent did not increase growth velocity. Betts et al. (1977) therefore concluded that decreased energy is a related factor to growth retardation in children with CRF, but not necessarily the only causal factor. Zinc deficiency has also been associated with growth failure in children (Prasad, 1961). In 1963, Prasad (1963), determined that the failure of male Egyptian youths to grow was due to a zinc deficiency. Subsequently, Walravens and Hambridge, (Walravens and Hambridge, 1976) demonstrated low hair and blood zinc levels in infants in the United States. With zinc supplementation, the male infants demonstrated improved body length and weight. The purpose of this investigation was to assess whether zinc acetate supplementation of children with End Stage Renal Disease (ESRD) improved energy intake and accelerated growth velocity and physical development. II. LITERATURE REVIEW A. Growth Introduction Growth and physical development of children are genetically predetermined. The normal rate of growth, however, can be influenced by such nutritional factors as undernutrition, overnutrition, or an imbalanced nutrient intake (Lowrey, 1978). Other factors influencing the growth and development of humans, both pre— and post—natally are thoroughly discussed in the treatise of Faulkner and Tanner, Human Growth, Vol. I,II,III, 1978—1979. Humans follow a normal progression of growth and development if provided with the appropriate environmental, physical, and social factors. Growth is the process of increasing in physical size while development is the gradual unfolding of the genetic potential of the organism. Anthropometry, radiography, and chemistry have been used to measure human growth and development. Tanner and thitehouse (1966, 1975, 1976) used physical measurements to (determine changes in height, weight, and somatic growth of ctiildren. Skeletal maturation through use of radiographs was sirudied by Gruelich and Pyle (1950, 1959). Macy (1942) followed changes in the chemical composition of blood and urine in a pediatric population. Standards have been developed from these early works (Tanner and Whitehouse, 1966, 1975, 1976; Gruelich and Pyle, 1950, 1959; Macy, 1942), and from subsequent studies (Hamill et al. 1979; National Research Council, 1980). These standards are now used to assess deviation from normalcy of an individual or group of individuals. Standards for Measuring Growth Anthropometric Robert's Nutrition Work With Children, (Martin, 1954) clearly summarizes the early standards developed to evaluate normal growth and development of children in America. Martin, (1954) credits Bowditch from the Harvard medical school with carrying out the first American study on height and weight in about 1872. According to Martin (1954), the Baldwin—Wood Weight—Height-Age Tables (1923) for boys and girls were the most complete and accepted tables in the early 1900's. In 1941, Dr. Norman C. Wetzel of Cleveland, Ohio developed the Wetzel Grid for evaluation of what he termed "physical fitness" (Wetzel, 1941). Wetzel (1941) Vliewed growth as a form of motion on three planes; quantity c>f growth, agents that control growth, and energetics of growth. Martin (1954) explains that physicians found this grid an effective screening device and it was generally used by health workers both in this country and abroad. The Stuart—Meredith Growth Tables of 1946 were developed from research conducted by each investigator individually: Stuart in Boston; Meredith in Iowa (Stuart and Meredith, 1946). This collaborative effort resulted in the use of five measurements to evaluate physical growth of school children and replaced the previous standards. Height, weight, chest Circumference, hip width, and leg (calf) girth were found to be reliable indicators of satisfactory growth. Percentile rankings for each measurement and each age were calculated and graphs developed. Meredith then in 1949 published the Meredith Physical Growth Record (Meredith, 1949). The Iowa Growth Charts for each sex were developed as a simplified form of the original data. They were to be used to assess the physical growth of Iowa school children. Recognizing the regional limitations of previous studies and the apparent gradual increase in the size of the American POpulation, the 84th Congress in 1956, authorized the National Health Survey. The studies resulting from this mandate consisted of five Health Examination Studies (HES) c0nducted through the National Center for Health Statistics (NCHS). Two of these health studies, HES IV and V introduced the specific focus of nutrition. These two studies are also known as the Health and Nutriton Examination Studies (HANES I and II). The NCHS growth charts were derived from data collected in HES II, III, IV, V and information from the Fels Research Institute, Yellow Springs, Ohio (Hamill et al, 1977). Charts for both males and females, ages 0—36 months, and 2—18 years were developed as smoothed percentile curves representing almost 70 million American children (Hamill et a1, 1979). A study group consisting of the American Academy of Pediatrics, the Maternal and Child Health Program, Public Hfiialth Service, and the Department of Health, Education, and wEilfare recommended that one set of data for all races would b6? sufficient for practical purposes (Hamill et al. 1977). Tfle NCHS charts are commonly used today as the best standards tC) evaluate the growth of American children regardless of ethnic origin or regionalization. In addition to height and weight standards, other anthropometric measurements have been developed to evaluate bOdy composition (Falkner and Tanner, 1978-1979). Among these are the triceps skinfold (TSF) to estimate body fat and the upper arm muscle circumference (AMC) to estimate lean bOdy mass. Seltzer et al. developed a reasonably precise method (r=0.795) for estimating the degree of obesity in Obese female adolescents using the TSF measurement (Seltzer et al. 1965). Durnin and Rahaman (1967) achieved approximately a 0.80 correlation coefficient between skinfold thickness and body density in a study on British adults and adolescents. Regression equations were calculated to predict body fat from the skinfold measurements with an error of about +/- 3.5%. Durnin's formulas (Durnin and Womersley, 1974) are widely used today in the evaluation of skinfold data. The Ten—State Nutrition Survey (TSNS), was approved by congress in 1967 (USDHEW, 1972). It provided the first comprehensive, single-source, life span data on body composition through the use of anthropometrics in the United States or any other country (Garn and Clark, 1975). HES cycle II data provided information on skinfold thickness of Children in the United States (Johnston et al. 1972; Johnston et al. 1974). Visweswara et al. (1970) studied the relationship of anthropometric measurements to nutritional status. Results Of their study indicated that anthropometric measurements, eSpecially the weight/height index were influenced by protein‘calorie malnutrition. Forbes and Amirhakimi's (1970) Work confirmed the data of other investigators showing that there is a linear relationship between skinfold thickness and body fat in normal children. Their data show that the average of six fatfold measurements obtained a higher correlation coefficient than the use of only one fatfold measurement (Forbes and Amirhakimi, 1970). Radiographs Roche (1978) credits Howard in 1928 with presenting the first radiographic data on the evaluation of growth through skeletal maturation. Baldwin et al. (1928), studied the bones of the hand, wrist, and lower forearm of children by means of roentgenograms to evaluate their anatomic growth. Gruelich and Pyle (1959) credit Todd who was director of The Brush Foundation, Cleveland, with gathering information on x- rays of the hand and publishing these data in 1937 in Todd's Atlas for Skeletal Maturity: I The Hand. This atlas was the predesessor to the standard atlas used today, Radiographic Atlas of Skeletal Development of the Hand and Wrist by Gruelich and Pyle (Gruelich and Pyle, 1959). Tanner and Whitehouse (1975) developed the TW2 (Tanner and Whitehouse 2) method to asses skeletal maturity. Depending on the level of nine maturity indicators or stages for each bone, numerical scores are assigned to 20 bones of the hand and wrist (Tanner and Whitehouse, 1975). Tanner et al. (1975) also developed a method for predicting adult height based on the TW2 method. Bayley's Bone Age (Bayley and Pinneau, 1952) was developed earlier to predict adult height and was based on the work of Gruelich and Pyle. The TSNS of 1967 also provided valuable normative data on the effects of nutritional status on ossification timing, bone remodeling, bone lengths, and bone proportions during growth (Garn and Clark, 1975). At the request of NCHS, Dr. S. Idell Pyle was asked to "assemble a standard of skeletal maturity from the Gruelich and Pyle Atlas for assessment of HES, cycle II radiographic data on children, aged 6—11 and 12—17 years" (Abraham et al, 1979). NCHS publications refer to this as the HES Standards (USDHEW, Series 11, no. 140, 1974 and no. 160, 1976). Dietary . It is generally accepted by many authors that growth is influenced by the nutritional status of the individual (Falkner and Tanner, 1978—1979; Martin, 1954; Lowrey, 1978;). Therefore, methodology relating to the collection and evaluation of nutrient intakes becomes important in the evaluation of growth. Burk and Pao (1976) summarized the evaluation of four quantitative dietary collection methods used in surveys of food intakes of individuals. The weighed record, estimated record, 24—hour recall, and the dietary history are compared uSing the following criteria: reliability relating to sampling and repeatability of data; validity; respondent burden; field survey costs; and data processing costs. 10 Validity is further subdivided in terms of being accurate (absence of systematic error), concurrent (two measures of same concept), construct (degree to which variability in concept is measured), and representative (measure on level taken as an indicator on more general level). The results of this comparison indicate that no one method was consistently advantageous. Therefore, Burk and Pao (1976) recommend that researchers decide which trade—offs are most relevant to their objectives. The TSNS was the first and most comprehensive attempt to Survey the dietary intakes of Americans at all age levels (Garn and Clark, 1975). Approximately 16,000 children Participated in the pediatric age group. A one day recall Was the method used in this survey. Results showed that "the absolute intakes of energy (calories) and of all the nutrients examined reflected socioeconomic status", (American Academy of Pediatrics, 1975) and that the quantity of food was the major dietary problem disclosed (Garn and Clark, 1975). HANES I was conducted between April, 1971 and June, 1974. This was the first program to collect measures of nutritional status for a scientifically designed sample representative of the U.S. civilian, noninstitutionalized Population in a broad range of ages, 1—74 years (Abraham et al. 1979). HANES II was started in February, 1976 and ended 11 in December, 1979. The focus of HANES II was to examine population subsets within the ages of 6 months to 74 years (Carroll et al. 1983). The Recommended Dietary Allowances (RDA) were first published in 1943 to "provide standards serving as a goal for good nutrition" (National Reseach Council, 1980). The RDA's were published in 1943 to determine how much manpower to direct to growing food to provide the necessary nutrients through food for a nation at war. The RDA's are now in their ninth revision and are based on the best scientific information available. RDA's are nutrient consumption recommendations for healthy population groups. The RDA's are recommendations that cover 95% of the nutrient needs for 95% of healthy people (National Research Council, 1980). Standards established to evaluate the HANES I dietary intake data were from a variety of sources. Iron, thiamine, and riboflavin were evaluated using standards from the 1968 recommendations of the Food and Nutrition Board, National Research Council while calcium and vitamin A standards were Closer to Food and Ariculture Orgainization/World Health Organization recommendations (Abraham et al. 1979). HANES data can now be used to compare intakes of subpopulation groups to the normative data for the total U.S. population (Abraham et al. 1979). B. Growth and Chronic Renal Failure Introduction The etiology of Chronic Renal Failure (CRF) in children is discussed by Boyer (1984). Broad categories presented by Boyer (1984) include: glomerulonephritis, pylonephritis and urinary tract malformations, renal dysplasia/hypoplasia, hereditary diseases, systemic diseases, vascular diseases, and miscellaneous other diseases (Boyer, 1984). The primary diseases leading to End Stage Renal Disease (ESRD) vary with age, Treatment for CRF varies somewhat with the primary diagnosds, the degree of renal insufficiency, and individual Variation (Boyer, 1984). Holliday outlines the progression of renal insufficiency (Holliday, 1976). Uremia occurs when the glomerular filtration rate (GFR) reaches a severity of 15—25 percent of nOrmal functioning mass (Holliday, 1976). With the onset of ESRD, artificial means of removing the waste products of mEtabolism become mandatory. Methods commonly used include hEmodialysis (HD), intermittant peritoneal dialysis (IPD), and continuous ambulatory peritoneal dialysis (CAPD). The InEthod of choice will depend upon dialysis center objectives alid individual patient variation (Tenckoff, 1974). Dialysis 313 indicated when the blood urea levels are greater than 150 13 mg/dl. and serum creatinine reaches 6-8 mg/dl. Blood values of patients with CRF are normally monitored to dertermine the extent of uremia and asymptomatic changes. Normail ranges of commonly monitored blood values as used at Children's Hospital of Michigan are given in Table 1. Medications and purpose of each medication commonly used to treat children with ESRD are listed in Table 2. Three of these medications, multivitamin preparation, folic acid, and RocaltrOl (vitamin‘Ds), are nutrient supplements necessary for either replacement of dialysate losses or replacement of Products normally metabolized in the kidney (Nelson and StOVer, 1984; Holliday et al. 1979). Amphojel and Dialume are trade names for aluminum hydroxide which is used in renal Patients for its phosphate binding properties. Side effects include a decreased absorption of vitamin A, possible oSteomalacia, constipation, anorexia, decreased weight, gastrointestinal cramps, a chalky taste and the inactivation 0f thiamin. Aldomet and Apresolene are both trade names for antihypertensives. Aldomet can result in drowsiness, dry mouth, diarrhea, edema, depression, nausea, and increased Weight. Hemolytic anemia can occur with an increased need f°r vitamin 312 and folic acid. An increase in serum alkaline phosphatase and creatinine, transaminase enzymes, and bilirubin are also seen with the use of Aldomet. Apresoline, can result in anorexia, gastrointestinal 14 distiress, dizziness, tremors, constipation, blood dyscrasias and aan increased need for pyridoxine. Phenobarbitol, an anticzonvulsant, can increase the turnover of vitamin D and K in cliildren resulting in rickets, decreased bone density, or osteomalacia. Other side effects of phenobarbitol may also include increased appetite, nausea, vomiting, and anorexia. Calcium carbonate, or Tums, is an antacid which can also be used as a calcium supplement. Calcium carbonate can result in infrequent hypercalcemia with alkalosis and decreased absorption of magnesium. Side effects of calcium carbonate include chalky taste, belching, nausea, constipation, and steatorrhea (Powers and Moore, 1983). The aims of appropriate dietary and pharmacological management are to (1) improve or stabilize renal function, (2) promote growth, and (3) permit the child to continue a normal life (Hollerman, 1979). Various reviews, (Fine and Gruskin, 1984; Pediatric Clinics of North America, 1979) are available on the nutritional and medical management of Children with CRF. In evaluating the growth in children with CRF, growth Velocity (cm/yr) and standard deviation score (SDS), (Falkner, 1962), also known as the Z-score (Snedecor and COChran, 1968), are usually used. Potter et al. (1978) recommend the use of the SDS because "normal" growth velocity is not known. These two methods of reporting results make 15 wmpalrison of data difficult. References on growth in childaren with CRF differ in the populations studied. Growth vehx:ity of children differs according to whether they have renal insufficiency, ESRD requiring dialysis, or kidney transplant. Growth Implications Children with CRF often have impaired statural growth (Potter and Greifer, 1978). West and Smith, (1956) in an attempt to elucidate the cause of growth retardation in renal disease, categorized the various factors into calorie insufficiency, chronic acidosis, or primary endocrine abnormality. Nash et al. (1972), studied a group of nine children With the presenting complaint of growth failure. Evaluation Of these children revealed an alkaline urine and renal tUbular acidosis of the proximal or distal type. In this Sample population, with alkali therapy, growth improved from below the third percentile for height age to the third to tenth percentile. Three patients achieved the twenty-fifth t0 fiftieth percentile for height. These patients were not aZOtemic. Metabolic acidosis may impair the biological activation of 1,25-dihydroxyvitamin D3 (Norman, 1982). Ten children with type I renal tubular acidosis, were studied for growth by McSherry (1978). Results of this study showed that 16 correction of the acidosis could be achieved with alkali therapy. The alkali requirement was sometimes in excess of 5 mEq of bicarbonate per kg of body weight per day. She concluded that with sustained correction, children will attain normal stature (McSerry, 1978). The oldest child in this study was 3.5 years with 7 of the ten children aged less than 4.5 months. Kohaut (1983), in a study on eleven patients undergoing CAPD for at least one year, showed a 0.274 POSitive, but weak, correlation between serum CO2 plotted against growth. However, the level of significance is not noted in the article. Patients in this study had a mean height growth velocity of 0.69 cm/month as compared with an expected velocity of 0.73 cm/month. Malnutrition will cause growth failure in otherwise normal children as well as children with CRF (Potter and Greifer, 1978). Malnutrition can be the result of inadequate energy, vitamins, and or minerals. Current dietary treatment for patients in renal failure is to decrease the dietary protein and maintain an adequate mineral intake based on measuring the blood mineral levels. Energy requirements are met by increasing carbohydrate and fat intake. Mnltivitamin supplementation, 1 tablet per day, plus 1 mg of f01ic acid is normally given (Holliday, 1979; Wassner,1982; Nolson et al. 1984). Simmons et al. (1971), and Betts and Magrath (1974) 17 established that adequate energy intake must be provided to achieve normal growth in uremic children. Simmons et al. (1971) determined that approximately 70 percent or more of the RDA for energy was compatible with growth in their patient population while Betts and Magrath (1974) determined that an energy intake of 80 percent of the United Kingdom's recommended allowances was required for normal growth to occur in their uremic patients. Work done by Boyer et al. (1974) demonstrated that a caloric intake meeting the RDA did not guarantee normal growth. In subsequent work done by Betts et al. (1977), 80 Percent of the energy recommendation for children in the United Kingdom, plus an additional 8.4% mean energy supplementation, did not increase growth velocity even though Some patients reported "improved well-being". These studies Suggest that decreased energy intake in children with CRF is a related factor but not necessarily the only factor in their growth retardation. CAPD patients obtain additional energy through the absorption of glucose from the dialysis solution. An investigation by Brown et al. (1973) demonstrated that 10 to 60 grams of glucose (40-240 kilocalories) per liter of dialysate may be absorbed during peritoneal dialysis. Likewise, De Santo et al. (1982) found that up to 140 gm/day (560 kilocalories) of glucose could be absorbed by children 18 during CAPD therapy while Moncrief et a1. (1985) cites higher amounts depending upon the number of exchanges and the concentration of each exchange daily. The amount of glucose absorbed is related to glucose concentration, absorption being almost complete within the first thirty minutes (Brown et al. 1973, Moncrief and Popovich, 1985). Both Arnold et al. (1983) and Takala (1982) used aggressive nutrition therapy to improve the nutritional Status and growth in renal patients. Arnold et al. (1983) worked with 12 children aged 2.5—11 years (9 males, 3 females) and demonstrated a 5.68 +/— 0.26 cm/yr growth Velocity with glucose polymer supplementation versus 3.90 +/- 0-58 cm/yr without supplementation. Each child served as hiS/her own control and was followed for one year both pre— and post-supplementation. All children were at least two Years prepubertal prior to initiation of the study. Arnold et al. (1983) concluded, however, that the nutritional deficit was small, but improved growth was seen when Supplementation was given to patients ingesting less than 75 Percent of RDA calories for height. Serum albumin Concentrations also increased significantly (p<0.05) which Provided additional evidence that a dietary deficiency was corrected. It has recently been reported (Kelley et al. 1984) that glucose polymers enhance the intestinal absorption 0f calcium in a liquid formula diet or in an aqueous 19 solution. Takala (1982) used total parenteral nutrition or continuous enteral nutrition to supplement three young renal patients (2 infants, one 4 year old). These patients had cessation of growth or deteriorating growth prior to the aggressive nutritional therapy. Results demonstrated a restoration of growth without an unreasonable urea production. Kleinknecht et al. (1980) followed 76 children with ESRD for more than one year to evaluate growth velocity and the relationship between bone length gain and bone maturation. Forty-five boys and 35 girls, aged 16 months to 16 years were followed for a mean of 29.6 months (boys) and 30.7 months (girls). A mean annual loss of length for age, compared to French national standards, was 0.4 SD/year for boys and 0.38 SD/year for girls. With dialysis, approximately one third of the patients showed a satisfactory growth rate (but no catch— up growth), one third had a moderately reduced growth rate and one third had severely impaired statural growth. The diet for this population was calculated to provide 75 percent of protein of RDA for children of similar height. Increased calories were provided using lipid and carbohydrate supplements. Kleinknecht et al. (1980) also demonstrated that the level of bone maturity was retarded for ctmonological age thus increasing the time span for statural garowth to occur. In boys the level of bone maturity for 20 puberty (a bone maturation score between 138-158) occurred at ages 15—18 years. In girls a bone maturation score of 134— 142 was reached between the ages of 14—16 years. Onset of puberty for normal children occurs at a bone maturation score of 130. This equates with a bone age of 13 for boys and 11 for girls. Therefore, it appears that the potential for growth is maintained throughout childhood and until pubertal growth and bone maturation occur. Kleinknecht et al. (1980) also concluded that growth velocity remained constant over several years of dialysis treatment and that children with the most retarded statural growth at the beginning of dialysis showed the best growth rate under dialysis treatment. Lowered serum creatinine levels were significantly related to improved growth. The techniques and schedules of dialysis have not previously been related to growth but Kleinknecht et al. (1980) state that the efficiency of dialysis and the accumulation of waste products, as demonstrated by serum creatinine, may play a role. Seventeen children aged 0.4—18.5 years receiving CAPD treatment, were studied by Stefanidis et al. (1983) for at least 5 months and compared to 18 children undergoing HD and 20 children who had received a renal transplant. Stefanidis et al. (1983) concluded that significantly (p<0.001) better growth was seen in patients treated by CAPD than in those reaceiving HD and that the treatment with CAPD did not show 21 significantly different growth rates from those of children who have kidney transplants. Other studies dealing specifically with CAPD provide additional information. Williams et al. (1983) concluded that patients on CAPD may be in longterm negative nitrogen balance if their protein intake is maintained at 1.0 g/kg/day or less. Balfe et al. (1981) studied 10 patients less than 6 years old and 9 patients older than 6 years for 6 months. These investigators demonstrated a growth rate of 0.31 cm per month with a range of O to 110 percent of absolute growth compared to predicted. Children less than 6 years of age also appear to lose greater amounts of protein (gm/kg/day), but absorb greater amounts of glucose (mg/d1) through the dialysate than children over 6 years. Salusky et al. (1982) evaluated the effect of CAPD therapy on children with CRF who had previously been treated by either HD or IPD. They found a mean growth velocity of 0.35 cm/month as compared to a normal growth rate of 0.5 cm/month in 9 patients, ages 1.5 to 11.4, on CAPD therapy for more than 6 months. They reported a significant decrease (p<.002) in mean serum total protein levels and a trend toward a decrease in serum albumin levels with the CAPD treatment. Energy intake was evaluated in twelve patients, including the 9 patients evaluated for growth velocity. Three of the 12 averaged less than 75 percent of the RDA 22 while the remaining 9 each had an average energy intake of 82 percent of the RDA for children of the same height and sex. They reported that the amount of energy absorbed through the dialysate was 7.8 kilocalories per kg per day. Data for a comparison of growth and energy intake for each subject were not presented. A more comprehensive evaluation on the nutritional status of 24 children undergoing CAPD was presented by Salusky et al. (1983). The patient population was divided into children less than 10 years (9 patients) and those 10 years and older (12 patients). Four younger children and 6 older children had a total energy intake (diet plus dialysate) of less than 75% of the prescribed intake (the RDA for energy and a minimum of 3.0g/kg/day of protein for children less than 5 yrs old, 2.5g/kg/day for 5 yrs to puberty, 2.0g/kg/day during puberty and 1.5g/kg/day thereafter). Growth, as determined by TSF, mid—arm circumference (MAC), height, and weight for chronological age and sex, was significantly reduced in the younger group, while the same was true for height and weight and MAC in the older age group. The MAC, however, was normal when evaluated against height per age. The TSF for the older group of subjects was normal. Serum total protein, albumin and transferrin were low and the amino acid concentrations were altered and similar to the pattern seen in children with 23 chronic uremia and malnutrition. A significant direct correlation was found between the TSF, expressed as Z—scores for chronological age (r=0.60,p<0.01) and height/age (r=0.70,p<0.001), and the duration of CAPD therapy. The authors conclude that these positive correlations indicate CAPD can promote "catch-up" accrual of body fat probably related to glucose absorbed from dialysate and that a weak correlation between Z—scores for MAC and duration of CAPD (r=0.43,P<0.05), supports the possibility that CAPD improves the nutritional status of pediatric ESRD patients. Continuous cycling peritoneal dialysis (CCPD) is a variation of CAPD (Brem and Toscano, 1984). CCPD is a combination of CAPD and IPD. Dialysis exchanges are made during the nighttime hours and a prolonged dwell is left in the abdomen during the day. Brem and Toscano (1984), compared growth in six patients on HD with 7 children maintained on CCPD. In the three patients on HD who had not reached puberty, linear growth averaged 3.5 +/— 1.1 cm/year, while in 6 of the pre—pubertal children maintained on CCPD, the growth velocity was 3.2 +/- 1.7 cm/year. Both groups ingested approximately two—thirds of the recommended age adjusted caloric intakes. Brem and Toscano (1984), concluded that no form of dialysis treatment holds any advantage over another in terms of growth in children with CRF. Delayed sexual maturation has also been shown to occur 24 in children with ESRD. Hutchings et al. (1966) describe a 15 year old girl, treated by hemodialysis, who had decreased growth and sexual maturation. Menstruation had not begun and there was no breast development or axillary hair. Likewise, Lirenman et al. (1968) present a case history of a boy Growth maintained on dialysis from 16 months to 16 years. was below normal and puberty was delayed for age. C. Growth, Renal Disease and Zinc Introduction Since the excretion of minerals in renal disease is altered, much attention has been given to monitoring the Na, K. Cl, Ca, and P levels. Little attention has been given to monitoring Zn levels. Prasad et al. (1963) established the relationship b’a‘lZWeen zinc deficiency and decreased growth in an adolescent Egthian male population. Walravens and Hambridge (1976) , and Hambridge et al. (1972), likewise, established that zinc deficiency was present in infants in the United States and Was associated with decreased growth. When there is a zinc deficiency, depressed taste acuity has been correlated with plasma levels of zinc in adults with CRF (Mahajan et al. 1980) . Anorexia is commonly observed in patients with CRF and leads to diminished energy intake (Blendis et al. 1981; Wassner, 1982; Holliday et al. 1979; Scharer and Gilli, 1984). Whether or not this anorexia is the result of changes in taste acuity has not been demonstrated. The factors affecting the absorption of zinc have not been c0mplete1y elucidated. Dietary phytates and fiber decrease the availabilty of zinc for absorption (Kelsay et 26 al. 1979), while the presence of dietary calcium enhances the binding of zinc with phytates (Prasad, 1978). Ronaghy et al. (1974) found increased growth in an adolescent male poptilation who were supplemented with zinc carbonate as mnnpared to a control non—supplemented group. The diets of bot}: groups were the same and contained unleavened wholemeal wheat bread rich in phytates. The location of zinc absorption is presumed to be the uplber jejunum (Matseshe et al. 1980). The absorption of zinc is thought to be aided by a zinc binding ligand (ZBL) (Evans et al. 1975). Zinc appears to be secreted into the jejunum af“ter stimulation from the pancreas by a meal (Matseshe et aJ-o 1980) and therefore must be reabsorbed to maintain pOSitive zinc balance. Once absorbed, zinc is transported by a transport Ptfiotein (Solomons, 1982). This transport protein is thought t<> be either albumin (Cousins, 1979) or transferrin (Evans, 19'26). Solomons (1982) presents an excellent review of the intrinsic and extrinsic factors regulating zinc absorption. AElmodt et al. (1981) found a 65 +/- 11% mean absorption in r“3rmal adult subjects using 10 micro Ci of carrier—free 65Zn administered orally after a 14 hour overnight fast. Istfan et al. (1983) studied the absorption of 70Zn in 4 healthy Sioung men and determined that the fractional absorption of ==inc varied directly with the stable isotope dosage given and 27 that this fractional absorption increased after restricted dietary zinc. Using 652m, Aamodt et al. (1982) studied the distribution, retention and excretion of zinc in 50 patients with taste and smell dysfunction. Results of this investigation revealed that 70% of the injected 65Zn activity was in the liver within five to seven days and was retained with a final biological half time (Tl/2) of about 75 days. The total distribution of zinc in the body was BOX-62% in Skeletal muscle, 20%—28% in bone, and 2%-4% in liver. The Same group of investigators (Babcock et al. 1982) evaluated the kinetics of oral zinc loading in this same group of Subjects. They determined that 10% of the total body zinc (2-1-2.5 g in normal subjects) was in a pool rapidly eXChanging with plasma and the remaining 90% was postulated to be in a single slow turnover pool which was probably Skeletal muscle. The kinetic curves which occur following administration of oral zinc loading could be accounted for by Changes in gastrointestinal absorption and renal excretion. In a study performed in rats by Jackson et al. (1982), Zinc dePletion was associated with a low plasma zinc level and an elevated plasma copper concentration. In these zinc deficient animals, bone zinc levels fell to 1/3 the normal 1eVel with zinc deficiency and remained significantly (p<0.001) below the normal values upon zinc repletion while 28 hair and skin zinc levels remained unchanged (Jackson et al. 1982). Jackson et al. (1982) also determined that the level of zinc in the testes of zinc-depleted animals was approximately half that of normal rats. McClain et al. (1984), working with rats, determined that both zinc deficiency and energy restriction produced hypogonadism and suggest that the hypogonadism in zinc-deficient animals is principally attributable to Leydig cell failure. In order to examine the relevance of the animal studies to man, Jackson et al. (1982) also measured zinc levels in a variety of tissues from three patients with disturbed zinc metabolism. The results of the investigation revealed decreased plasma, hNIt normal hair, skin and muscle zinc levels. Post mortem Zinc levels were analyzed on tissues from one patient. ReSults showed low zinc levels in bone, liver, and testes. Zinc is excreted from the body primarily via the feces. SeCondary routes include urine and sweat (Underwood, 1977). In a balance study on humans performed by Milne et al. (1933): it was determined that 0.4—0.5 mg of zinc per day cOuld be lost through sweat. The zinc lost via sweat was reduced by 50 percent or more when the amount of zinc given Was reduced from a mean of 8.3 mg/day to 3.6 mg/day. ResearCh on zinc has also determined that renal tubular disease leads to the failure of tubular reabsorption of zinc (Beisel et a1. 1978). During catabolic states, the excretion «(was marr- v‘ WW" 29 of zinc in the urine has been related to loss of muscle tissue (Kirchgessner et al. 1978). Zinc is a component of hundreds of enzymes and other proteins. In animals, the alkaline phosphatase activity in serum declines rapidly as a result of zinc deficiency. Bone and kidney alkaline phosphatase activity may also decline vvith zinc depletion (Kirchgessner et al. 1978). Growth and Zinc Studies with rats demonstrated that zinc is essential fOr normal fetal growth (Hurley et al. 1966; Warkany et al. 1972), tissue development (McKenzie et al. 1975), and maternal weight gain (Fosmire et al. 1977). Prasad et al. (1963) published a classic study associating zinc deficiency with growth in a male Egyptian adolescent population. Hambridge et al. (1972) later reported poor growth, hypogeusia, and zinc deficiency in an otherwise normal child population in Denver, Colorado. Walravens and Hambridge (1976) measured weight, height, and head circumference in a healthy zinc supplemented and control infant Population. Results of this investigation indicated a significant increase in the supplemented group for all three Parameters in male infants. During clinic visits, parents of all sublects were asked if their infants experienced ConStiPation, diarrhea, or vomiting. The zinc supplemented 30 group reported significantly decreased gastrointestinal problems (p<0.005). Energy intake was the same in both groups of infants studied. In 1983, Walravens et al. (1983), supplemented a group of low income children from Denver with zinc and compared their linear growth with controls from the same population pair-matched, for sex, age, and initial height for age percentiles. These children had low growth profiles and were Presumed to be zinc deficient. During the first 6 months of the study there was no significant difference between test and Control subjects although a trend for a greater height increment was seen in the zinc supplemented boys compared to their pair—matched controls. However, at the end of 12 months there was a significant difference in height growth Velocity between test and control groups and these differences were primarily due to the increase in height gain of Zinc Supplemented males. It was also noted in this study that energy and protein intakes did not differ significantly between the groups upon entry into the study, but were Significantly greater in the zinc—supplemented group at the end of the study period. Zinc and Renal Disease Growth retardation is documented in zinc deficient Children (Prasad et al. 1963; Hambridge et al. 1972) and in 31 children with CRF (West and Smith. 1956; Potter and Greifer, 1978). Low plasma zinc levels and significantly depressed RBC zinc (p<0.05) have also been documented in children with CRF (Siegler et al. 1981). In a comparison of three adult hospital dialysis programs in Toronto, Canada (Blendis et al. 1981), there was a significant difference in plasma zinc levels between patients in hopsital A and hospitals B and C patients. The estimated zinc and protein intakes were Significantly greater from patients from hosptial A than from patients from the other two hospitals. These authors cOnCluded that the zinc deficiency seen in hospitals B and C ‘Vas due to a low protein diet in their chronic dialysis Ekatients. Hypogeusia was implied as a possible cause of the decreased protein intake. Slight leaching of the dialysis eqUiPment was noted as a possible reason for combating the develOpment of zinc deficiency in hospital A patients. Likewise, Mansouri et al. (1970) noted that undialysed children with CRF on a low protein diet had lower plasma zinc leV81S than dialysed uremic subjects on a normal protein intake. Although not discussed by Mansouri et al. (1970), the lower plasma zinc in the nondialyzed subjects may well be due to the dilution effect caused by the edema of CRF. Fourteen patients with CRF were evaluated by Siegler et a1_ (1981) for zinc deficiency. Results of this study indicated a strong correlation (r=0.96, p<0.01) between both 32 energy and zinc intake and protein and zinc intake. A similar correlation was found between erythrocyte zinc and both percent ideal body weight (r=0.56, p<0.05) and height percentile (r=0.61, p<0.01). D. Analysis of Zinc Zinc can be analyzed in biological fluids by various techniques. According to Henry et al. (1974), Clinical Chemistry: Principles and Techniques, the method of choice is based upon many factors. These factors include the sensitivity of the method, reproducibility, precision or accuracy, analytical recovery, sample size required, Simplicity or convenience of the method, and repeatability. The conventional method for the analysis of zinc in blOQd is atomic absorption spectrophotometry (Walsh, 1955). Prasad et al. (1965), described a technique for the dEtermination of zinc in plasma, red blood cells and urine Using a commercially available spectrophotometer. Prasad et a1. (1965), used this technique to determine the red blood Cell: and urine zinc levels in patients with cirrhosis of the liver and compared their results with the dithizone method. COmparable results were obtained. In addition, atomic absorption spectrophotometry, was much simpler, less time Consuming, and more sensitive than the dithizone method. Curtis and Roth (1978) define atomic absorption as the determination of "the type and amount of an element by measuring wavelength and intensity of the light absorption of lts mono—atomic vapor". Conversion of the element to atomic 34 vapor from a solution or solid is generally carried out using a flame. The heat from the flame nebulizes the liquid, evaporates the solvent and the dissolved particles, and causes atomic dissociation of the molecules of salts in the vapor phase. Atomization occurs. In atomization, heat excites the electrons to raise from their ground state or orbital rings. When under low temperature, heat is released, but when using a higher temperature, light, which can be measured by absorbance, is released. Even a higher temperature will ionize the substance and remove it from the medium. Therefore, an optimal temperature for each element is required. Various modified techniques of the atomic absorption mathod are noted in the literature. Matsuura (1982) cornpares flame and flameless atomic absorption techniques and Concludes that the flameless as compared to the flame requires a smaller specimen and has much higher sensitivity, but requires much more elaborate preparation and does not have good reproducibility. Sekiya et al. (1981) and whitehouse et al (1982) concur. Foote and Delves (1982) ~btained a 0.94 correlation coeff1c1ent between flame atomic absorption and electrothermal atomization techniques. The primary advantage of electrothermal atomization over flame a . tQmic absorption is the much smaller sample required. This :1 % particularly useful in measurements made on biological 35 fluids or tissues of neonates and newborns. Taylor and (1981) compared sample pretreatment methods using water, butan—l—ol or propan—1~ol, None of these methods showed e1 e ctrothermal atomization . and Bryant trichloroacetic acid, superior between-batch precision. The inclusion of sodium and potassium ions was also examined by these authors. Flow Ionization interference was found to occur. injection analysis was shown by Rocks et al. (1982) to offer direct analysis of copper and zinc in small volumes of fast , Discrete nebulization of a sample as compared to serum. conventional nebulization was reported by Makino et al. (1981) to have slightly poorer precision. D III. METHODS AND PROCEDURES A. Research Site Children's Hospital of Michigan is a 290 bed teaching hospital located in Detroit, Michigan. The hospital is affiliated with Wayne State University School of Medicine. Children between the ages of birth and 18 years are routinely treated for renal diseases, including ESRD. Dialysis facilities exist for both hospital based HD and IPD therapy, and CAPD treatment for outpatients. Hospital support services include laboratory facilities for routine chemical analyses, radiology services for obtaining and reading hand WI‘ie:t radiographs, and a professional nephrology staff who COmmonly participate in research projects. Dr. Larry Fleischmann who is Director of Nephrology and all dialysis programs at Children's Hospital of Michigan, actively pa:E‘ticipated in this study. B. Subjects All patients, aged 6 to 18 years, who regularly undergo I‘§atment for ESRD at Children's Hospital of Michigan, Q"Iroit, Michigan, were invited to participate in the study. 37 The project was approved by the Research Grants and Investigation Committee at Children's Hospital of Michigan and the University Committee on Research Involving Human Subjects (UCRIHS) at Michigan State University and subsequently has been renewed annually. Signed consent forms (Appendix A) were obtained from all legal guardians prior to initiation of the study and/or assent obtained from patients 12 years and older. All analyses not done at the hospital for clinical purposes were completed using the medical record number to assure confidentiality of the subject. Subjects were able to withdraw from the study at any time. All patients were diagnosed as having ESRD and were baing treated for their renal failure by hemodialysis (HD), intermittant peritoneal dialysis (IPD), or continuous ambulatory peritoneal dialysis (CAPD). Patients were changed from one method of treatment to another as determined by the Clinical judgment of the attending physician. During the couIli‘se of the study, 26 patients participated. 0f the 26 Su‘bjects, 17 were male and 9 female, while 11 were Black, 14 white and 1 Asian. The characteristics of the study pa1“ticipants are given in Table 3. Patients treated for their ESRD by CAPD or their Qal‘etaker, received an extensive 2 to 3 week in 1mglise training course. Each member of the nephology team phavided training in their area of specialization and was 38 required to sign off as to the competency of the patient before home care was approved. The dietary training was given by a masters prepared registered dietitian. Therefore, the participants in this study were well prepared to provide dietary intake data, and comply with all medical instructions as well as zinc supplementation instructions. 0. Dialysis Fluids Hemodialysis patients were dialyzed for approximately 3 hours, 3 times each week. Eri-lyte 8306 hemodialysis concentrate (Erika Inc., Rockleigh, N.J. 0764?) was used in a Drakes Willock Dialyzer (Portland, Oregon) to ultrafiltrate Patients' blood. The composition of the dialysis fluid is given in Table 4. Children maintained on CAPD used Dianial PD‘2 manufactured by Baxter Travenol Laboratories, Inc (Deerfield, Il.) to complete 4 to 5 exchanges daily. In CAPD, dialysate is gravity fed into the peritoneal cavity where it dwells for two or more hours depending upon the irlciividual patient's schedule. The composition of Dianial PtL2 dialysate is listed in Table 4. Dianial PD—2 is bgtassium free as are solutions used in IPD and HD. However, the dextrose concentration can be 1.50, 2.50, 4.25 gm G‘throse per 100 ml. Higher dextrose concentrations increase 1:1'1e osmolarity of the dialysate thus removing larger 39 quantities of water from the patient. Patients are evaluated monthly by the attending physician and dialysis staff and the appropriate solutions and schedules are prescribed. A combination of different solutions could be used for separate exchanges during the course of the day. CAPD patients are home trained on the maintenance dialysis treatment and provide their own care or the care is provided by a trained Patients maintained on IPD are caretaker, usually a parent. dialyzed for approximately 12 hours, 3 times per week using a AMP 80/2 Peritoneal Dialysis System (Freehold, N.J. 07728). Hemodialysis and IPD patients receive dialysis treatment at the hospital where specialized equipment and staff are available . D. Anthropometric Measurements Anthropometric measurements included height, weight, MAC . and TSF. Measurements were usually obtained monthly at regularly scheduled clinic visits according to the method dfiscribed by Foman (1976) . Height was measured on each Subject undergoing hemodialysis using a non—stretchable tape affixed to a true vertical surface in the dialysis suite. A r"la-nual headboard was constructed by the maintenance staff at QI‘lildren's Hospital of Michigan. This headboard was placed 911 the head of each subject while standing against the 40 vertical metal tape with their heels, buttocks, shoulders and head against the wall and with the head held parallel to the floor, facing forward (Foman, 1976) . Markings were made at the base of the headboard and the measurement was read in cm directly from the tape. Height measurements on subjects undergoing CAPD treatment were obtained at the beginning, the initiation of zinc supplementation, and end of the study by the principal investigator using a calibrated digital counter stadometer (Holtain Limited, Crymych, England). Monthly heights were taken by nursing personnel regularly assigned to outpatient duty. A stadometer (Continental Scale Corporation, Bridgeview, Ill. or Detecto Scales Inc, Brooklyn, N.Y.) was used. The prediction of adult height was determined by calculating the midparent stature and by using the method of BaYley and Pinneau (1952) . Calculations based on the work of Garn and Rohmann (1966) were also completed to determine the C“ll‘li‘ent extent of growth necessary to achieve midparent helight. Body weights were measured to 0.1 kg on either a cQl'ltinen’cal or Detecto beam balance (Continental Scale Q0I‘poration, Bridgeview, Il.; Detecto Scales Inc, Brooklyn, N ‘ Y.). Beam balances were commercially calibrated several tines per year. The Detecto beam balance in the dialysis $11:ite was documented as being calibrated 4 times in 1984. ¥ .4 41 Nursing personnel were responsible for collecting weights on all CAPD children and patients when hospitalized. Hemodialysis and IPD patients were taught to obtain their own weight pre— and post—dialysis treatment. Post—dialysis weights were used in the study for hemodialysis subjects. Weights on CAPD subjects were obtained after subjects had emptied their dialysis bags. MAC and TSF measurements were taken by the principal investigator. MAC was obtained using an Inser—Tape, compliments of Ross Laboratories (Ross Laboratories, Columbus, Ohio 43216). The midpoint was determined using a Dritz (John Dritz & Sons, Inc. N.Y., N.Y.) non—stretchable tape. Lange calipers (Cambridge Scientific Industries, Inc., Cambridge, Maryland 21613) were used to measure TSF. The Lange caliper was calibrated frequently using the metal cal ibration standard supplied with the instrument. E. Radiographs Radiographs of the hand and wrist were taken as part of a regular skeletal survey prior to zinc supplementation by radiology technicians at Children's Hospital of Michigan. At the completion of the zinc supplemented period, a second 3:la-nd-wrist x-ray was taken. Additional baseline radiographs wfire available on 5 subjects so that change over time was 42 compared between pre—zinc bone age and post—zinc bone age. All radiographs were assessed for bone age according to the method of Gruelich and Pyle (1959), by Thomas Slovis, M.D., pediatric radiologist at Children's Hospital of Michigan. F . Physical Examination A physical examination was obtained by the attending physician and/or the clinical nurse specialist at least monthly throughout the study. The purpose was to provide ongoing medical care. CAPD patients were examined approximately every month as outpatients, unless hospitalized, in which case more frequent examinations were nOted in the medical record. Physical examination data were uSed by the principal investigator to track medical and/or sC’¢ial events, such as infections, changes in treatments, compliance to medical instructions, and other disease CQnditions which might influence the results. Tanner staging (Tanner and Whitehouse, 1976) , to d‘etermine the degree of sexual maturation, was completed d‘3lI‘ing the physical examination both before and after zinc s‘lpplementation by the attending physician or the clinical nurse specialist. Developmental changes in the male Q-Qnitalia and pubic hair growth in males, and breast and DIlbic hair growth in girls, provided an index to evaluate the 43 influence of puberty on statural growth. G. Laboratory Analyses Laboratory data commonly obtained for routine care of renal patients included blood determinations for calcium, phosphorus, sodium, potassium, chloride, magnesium, urea nitrogen, creatinine, alkaline phosphatase, total protein, hemoglobin, hematocrit, 002. Blood samples were collected monthly, either at the time of the clinic appointment or prior to the initiation of HD, which was usually in the morning after breakfast. The Centrifi—Chem analizer (Union Carbide Corporation, RYE, N.Y. 10580) was used to measure alkaline phosphatase by a nlocilified Bessey, Lowry, and Brock technique (Bessey et al, 194'5) . and serum creatinine using alkaline picrate. The centI'ifi—Chem is an automated instrument and is also used to measure magnesium (Grindler and Heth, 1971). Eastman Kodak 's Ectachem equipment (Rochester, N.Y.) was used to determine blood urea nitrogen using a urease reaction; serum chloride, sodium, potassium and 002 using an ion specific ElectIrode; serum phosphorus using a Fiske Subbarow molydate reduction reaction; serum calcium using an arsenazo-S dye COmp . . lex; total protein uSing the biuret reaction; and serum 31 1:>“1“‘lin using bromcresol green color reaction (Eastman Kodak, 44 1985) . A manual method using ammonium hydroxide was used to determine hemoglobin, while the hematocrit was measured manually by centrifugation. These values were recorded in the patients medical record and were used to evaluate the severity of renal failure and to monitor dietary compliance. The diet prescription was readjusted by the attending physician when necessary as indicated by the laboratory values. H. Zinc and Copper Analysis Erythrocyte and plasma zinc and copper levels were determined by atomic absorption spectrophotometry (AAS) , both pre— and post-supplementation, in the laboratory of Dr. Prasad, Veterans Administration Hospital, Allen Park, Michigan. This method has previously been published by Prasad et al. (1965). Blood samples were collected both pre— and post— Su«”39lementation by nursing or laboratory personnel at Children's Hospital of Michigan. A 5—10 ml sample of whole blood was drawn with a needle and syringe and put into a Nalge (Nalgene Labware, Rochester, N.Y.) test tube containing erps of zinc free heparin. Extra care was taken to prevent contamination of the sample. The sample was capped, the blood “‘1 Red, and refrigerated. After 2 to 4 hours, 45 sample was centrifuged (Sorvall RCZ-B) at 3000 rpm for 20 min at 4° C. The supernatant (plasma) was transferred to a second Nalge tube, the buffy coat discarded by pipetting, and the precipitate (RBC) washed with 0.996 cold saline (NaCl + deionized H20) . The erythrocytes were centrifuged again at 3000 rpm for 20 minutes at 4° C. The supernatant was removed by pipetting and discarded. This washing of the erythrocytes was repeated 3 times. One ml of 1096 trichloroacetic acid (TCA) was added to 1 m1 of plasma to precipitate the plasma proteins. The plasma then heated for 10 min in a 110° water sample was vortexed, The bath, and centrifuged for 15 min (3000 rpm) at 15—-20O C. supernatant was then poured into a clean Nalge test tube and the process repeated two more times on the precipitate. The SupeI‘natant each time was added to the previous collection. The total collection was brought to 3 ml with deionized H20 and Was analyzed for zinc and copper concentration. Erythrocyte zinc was determined by pipetting 0.5 ml of packed RBC's using a plastic serological pipette into an 18 m ‘1 Nalge test tube. One ml of deionized H20 were added using an all‘tomatic pipetter, the sample vortexed, and 1 ml of concentratEd HN03 added using a repipettor attached to a HN03 bottle. The sample was lightly capped, placed under a fume hood in a 110° hot water bath, uncapped, and heated for 2 to 2 The volume of the sample was brought to 4 ml 1 / 2 hours. 46 with deionized H20 by pouring the sample into a graduated glass test tube, rinsing the Nalge test tube with deionized H20, adding this rinse to the sample, and finally adding more deionized H20 to 4 ml. The sample was then decanted back into the original Nagle test tube and zinc and copper concentrations measured. Standard solutions were made using zinc oxide (Fisher Scientific, Pittsburgh, Pa 15219) and copper nitrate (Fisher Pittsburgh, Pa 15219) in dilute nitric acid. The Scientific, ccuncentrated standard of 100 mcg/ml was made fresh each month ‘byr diluting 3 times with deionized H20 to obtain the final ccuicentration range of 0.2ug/ml to 1.0 ug/ml. A Perkin Elmer 306 Atomic Absorption Spectrophotometer was used to measure the concentration of zinc and copper in standard solutions, and plasma and erythrocyte preparations. The spectrophoto- meter wavelength was set at 214 nm and 325 nm for zinc and coPper analyses, repectively. I. Food Intakes A dietary history and a 24-hour dietary recall were ' c: hrtaiined on each subject at the beginning of the study using <3 :1E3‘3ary department forms (Appendix A). Each participant or I1 is/1'1er caretaker was requested to keep a 3 day (2 weekdays is fit: 1 weekend day) food diary of all food ingested at the 47 initiation of the study and for each month throughout the study. These records were then reviewed with the subject or caretaker by the clinical dietitian to expand and clarify information. Frequently, during periods of hositalization, food records were collected by hospital staff to monitor and fluid intake. 1980; USDA, energy, protein, sodium, potassium, Using standard references (Pennington et al, 1975), calculations for energy, protein, sodium, and potassium intakes were made of the 3 day food record, 24—hour and food intakes, to determine the diet prescription Energy and protein levels were recall, for each participant . prescribed according to those recommended for age. Specialized supplements such as Polycose (Ross Laboratories, Columbus, Ohio), Propac (Biosearch, Somerville, N.J.), and Amin—Aid (American McGraw, Irvine, Ca) were prescribed and encouraged if the manually derived calculations indicated a need for additional energy or protein. The 3 day food I'e‘130rds, 24—hour recalls, and hospital food records were then coded for nutrient analyses using the Michigan State University computerized nutrient data base (Morgan and Zabik, 1983). Each subject's daily intake was evaluated as percent 0 f IRDA for age and per weight for energy and protein, and the R DA per age for calcium, phosphorus, sodium, chloride, Do . . . . tQSSium, magnesium, ZlnC, copper, and iron. Since subjects were not consistent in bringing in the 3 48 day food records, the intake data (24-hr recall, 3 day record, or hospital food intake) from the one date closest to the end of each month were used for statistical evaluation of each subject's dietary intake. The mean values were compared to the HANES I data for children of the same age and sex for energy, protein, calcium, and iron. HANES II standards were used to compare each subject's mean data for energy, protein, calcium, phosphorus, iron, sodium, and potassium with American children of the same age and sex. Information provided in the HANES I data allowed for Z-score analysis of each subject's data. However, the information provided in HANES I was limited to only energy, protein, calcium, and iron. HANES II data contained a more complete listing of nutrients, but did not provide standard deviations for the Amarican population thus negating Z-score calculations. Therefore, each subject's intake for energy, protein, CalCtium, phosphorus, iron, sodium, and potassium was calczulated as a percent of the 50th percent of HANES II data, to see if there were significant changes in intake bet‘Meen the non—zinc supplemented and zinc supplemented peI‘llods. 49 J. Zinc Supplement A liquid form of zinc acetate (USP Reagent) was prepared in the pharmacy at Children's Hospital of Michigan by the investigator. Using a Mettler balance, 56.1 gms of USP Reagent grade zinc acetate (Frank W. Kerr Chemical Co. , Novi, Michigan) was weighed. The powder was then added to 1000 ml of sterile water (Travenol Laboratories, Deerfield, 11.) and mixed by shaking in the original container. The final concentration of the mixture was calculated to be 20 mg of elemental zinc per ml. The zinc acetate solution was. poured into a 2 or 3 oz amber bottle and labelled. Each bottle contained sufficient zinc acetate solution to last one month Therefore, zinc acetate was provided on a monthly basis to eaCh subject. The amount of zinc supplementation for each subject was calculated based on the recommendations of Walravens (1979) and through personal communications with Dr. Prasad. The quantity was determined by multiplying the patient's body w e eight in kilograms by 2 mg/kg. The total amount prescribed W as the result of this calculation, but in no instance e Xceeded a total of 40 mg of elemental zinc per day. Bottles O f liquid zinc acetate were labelled with the patient's name, t he name and telephone numbers of Dr. L. Fleischmann and 50 Dorothy Hagan, the concentration of the solution (5.6 gm/100 ml), the amount delivered per ml (20 mg elemental zinc per ml), the prescribed prescription for the subject, administering instructions (Take ml daily, 1 hr. before breakfast) and the current date. Participants and/or caretakers were verbally instructed and shown how to measure the zinc acetate liquid using an oral syringe (Baxa, Denver,Co) with adaptor. The prescribed dosage level was marked on the syringe for the convenience of the subject or caretaker. Zinc supplementation was started after collecting one year of baseline data on all except 2 subjects. At least 3 The months of data were collected on these 2 exceptions. subjects were maintained on zinc supplementation for one Year (April, 1984 to April, 1985). K. Statistical Procedures Statistical procedures used to evaluate the data "Included calculations of means, standard deviations, Z- s . cOres, correlation coeffiCient, and the student t test (Snedecor and Cochran, 1968) . Ages for the study population w I n are converted to decimal age (Buckler, 1979) to Simplify In athematical manipulation of data. Growth was evaluated using a variety of procedures. A 51 correlation coefficient between height and decimal age was determined for each subject both before and after zinc acetate supplementation. The student t test was then used to determine the level of significance. Mean and standard deviations were calculated on growth velocity (cm/yr) data for male and female subjects. Growth was further evaluated as height per age and weight per age. Calculations of 2— scores were made to show change between the beginning of the study, the initiation of zinc supplementation, and the end of the study. Correlation coefficients for each subject were calculated between the height per age, weight per age, and weight per height both before and after zinc acetate supplementation. The student t test was again used to datermine if the correlation coefficients were significant. PerCent medians and Z—scores were calculated on AMC, and TSF, using HANES I data (Johnson et al, 1981; MAC . respectively , Johnston et al, 1974) as the standard. Bone maturation was c°mpared to HANES I standards (Roche et al, 1974; Roche et a1 ' 1976) and Z—scores determined. The deviation from the pr'edicted adult height for each subject was evaluated using a Z\ SCtore. Mean and standard deviations were calculated for each s ubject for all biochemical parameters both before and after 2 . 111C acetate supplementaion. Statistical significance b etween the pre— and post—supplemented values was determined. L‘ _4‘. 52 Mean and standard deviations were calculated for each subject for dietary intakes both before and after zinc acetate supplementation. Using HANES I and II data for age and sex (Abraham et al. 1979; Carroll et al. 1983), these dietary intake data were then evaluated using Z-scores and percent median, respectively, for each subject. The student t test was used to test for significance between mean values on each subject. IV. RESULTS A. Introduction Of the 26 participants in this investigation, 9 completed the study, 4 males and 5 females. Three additional male subjects completed the study, but refused the zinc supplement and could only be evaluated for the non—zinc supplemented phase of the study. Thus a total of 12 subjects 7 males and 5 females, were followed before zinc acetate :supplementation and 4 males and 5 females after :supplementation. The remaining subjects were lost to the sstudy for the following reasons: 7 transplant, 2 death, 2 Huoved from the area or transferred to another health faicility, 2 withdrew, and 1 could not be followed (Table 3). The primary diagnosis and daily urine output for each pearticipant are listed in Table 5. Six of the 12 Pairticipants were anephric, 4 produced 0—50 cc of urine per dEiyn and the remaining 3 had urine outputs of less than 1000 CC per day . The initial heights and weights of the 12 subjects who CEDrnpleted the study are shown in Table 6. Ages of the SL1bjects were quite varied. Male ages ranged from 5.7 to 151-5 decimal years at the initiation of the study. For the 5 k 54 females the decimal age range was 5.2 to 19.6 years. The prediction of adult height based on two methods, midparent stature and the method of Bayley and Pinneau (1952), are presented in Table 7. Predictions are given for children completing the study and are based on height 2. measurements obtained at the end of the study period. scores calculated from the predicted adult height and HANES I (Johnson et al. 1981; Johnston et al. 1974) mean adult height show all subjects will be below the mean adult height. Predicted height for 2 older subjects, nos. 14 and 26, were less than 1 standard deviation below the adult mean height of 25 year olds and 5 other subjects (nos. 17, 15, 18, 19, 24) varied from the adult mean by more than 1 standard deviation. IPredicted heights for 2 subjects, nos. 10 and 9 varied from 'the adult mean by more than 2 standard deviations. Based on :Einal height measurements, the height the child should have Eichieved per chronological age to ultimately reach the nuidparent height (Garn and Rohmann, 1966) are listed in Column 6, Table 7. B. Growth Measurements Growth velocity calculations expressed in centimeters PEEI‘ year (cm/yr) for each subject when non-zinc supplemented a11<>pulation varied from the mean for their age and sex by 2 t0 CD‘Ier 6 standard deViations, regardless of zinc SIlpplementation. Only two subjects (nos. 18, 26) (iennonstrated deviations less than 2 from the mean for height fCDI' their age and sex. Five subjects moved closer to the mEEEin with zinc supplementation, 5 moved farther from the mean 56 with zinc supplementation, and 2 remained essentially at the same distance from the mean for their age and sex. Weight per age for both male and female subjects is shown in Table 10 as Z-scores. Distance from the mean weights of the American population show all subjects, except subject no. 18, were below the mean for their age group and sex. Individual data show equal movement of the number of subjects to and from the mean pre- versus post—zinc supplementation. Weight for height comparisons are an indicator of malnutrition. Scharer and Gilli (1984) recommend standard deviation score comparisons for height and weight be used so- that height, weight, and age can be included in the evaluation. However, reference data were not available to (calculate weight per height Z—scores. Therefore, a czomparison of the Z-scores for height per age and weight per aage is presented together in Table 10. In most subjects, the ZZ—score for weight is approximately half the Z—score for fleight. This indicates that weight was closer to the mean Of ‘tlie American population than was height, and that the weight Per height was adequate. Significant correlation coefficients for height per age, Weeight per age, and weight per height, based on individual Péitient data, are presented in Table 11. Nineteen Sixgnificant correlations were found prior to zinc $11Ibplementation while only 6 were found after zinc ; 57 supplementation. Height per age was significant for subjects no. 5, 17, 15, 1, 14, 18, 19, and 23 before zinc supplementation, and only subjects no. 5, 17, and 24 after supplementation. Significant correlation coefficients for weight per age, were found for patients 5, 17, 18, 23, 24, and 26 prior to zinc supplementation, and subjects no. 5, 17, and 24 after zinc supplementation. A significant correlation coefficient for weight per height was found for subjects no. t 5, 17, 15, 18, and 23 in the non—zinc supplemented group and only subject no. 5 in the zinc supplemented group. Weight per height, when standards were available for calculation, are presented as percent median in Table 12. All subjects for whom reference data were available demonstrated éadequate weight for their height. Missing calculations :indicate that the height per age value per subject was below tile available HANES I reference standards for height—weight- age tables . C. Anthropometric Measurements Mid-arm circumference, TSF, and AMC data for each SIiject are presented in Appendix B. Arm muscle cizrcumference is presented as percent median while MAC and TESF‘ are calculated as Z—scores (Table 13). These data ir10:1icate AMC values close to the median for age and sex while g __....u 58 MAC and TSF values show a negative deviation from the mean in all but 2 cases during both period I (before zinc supplementation) and period II (after zinc supplementation of 9 of 12 subjects). However, the TSF hovered around the mean in all cases except 3 subjects in period I, and 3 subjects in period II. Only 1 of these 3 subjects (no.24) in period II received zinc supplement and showed a negative standard deviation of 1.04. Subject no. 14 who did not receive zinc supplement had a higher negative standard deviation of 1.33 while the other zinc supplemented subject (no. 18) showed a positive standard deviation of 1.98. MAC varied from the mean by more than 1 standard deviation in 7 cases in period I tand 7 cases in period II. Zinc supplemented subjects nos. 5, 10, 17, 18, and 24 moved closer to the mean for MAC while suibjects nos. 5, 18, 19, and 23 showed a more positive TSF, 53eriod I versus period II. This positive movement in 4 srubjects (nos. 5, 18, 19, 23) for TSF may indicate an ixicrease in fat stores when zinc supplemented, but is c<3untered by the fact that 2 of 3 non—zinc supplmented cliildren also showed improved Z—scores during period II. In 7 out of 9 children supplemented with zinc, there was an irlcrease in the percent median for AMC, while 1 of 3 non—zinc Sllxbplemented subjects improved his percent median for AMC. 59 D. Chronological Age and Bone Age Comparisons The chronological decimal age at the time radiographs of the hand and wrist were taken for males and females and bone age readings are presented in Tables 14 and 15. Bone age percentage of chronological age and Z-scores are given for male and female subjects at the beginning of the study, at the initiation of supplementation and at the end of the zinc supplemented period. The bone age of the 7 male subjects, prior to zinc supplementation ranged from 0.2 — 5.3 years less than the chronological age or from 40 - 95% of chronological age. I?or the child 4.2 decimal years, the age was only 0.2 years taehind. This subject, no.1, was the youngest subject and Vvas 95 percent of chronological age. In 3 of the 4 male and 2 of the 3 female subjects (n05- ‘5 , 17, 15, 18, 9), who were zinc supplemented and for whom (fiesta were available, there was a positive change in the EDEBrcent median with zinc acetate supplementation. One of the 3 non—supplemented male subjects also showed a positive Cilange in percent median during similar time periods, while 011E: subject remained unchanged and one showed a negative Clieinge. Similar changes were seen in the Z—score CEilculations. Three of the 4 male subjects (nos. 5, 10, 15) L i 60 and 1 of the female subjects (no.18) showed a positive movement toward the mean with zinc supplementation. However, 2 non-zinc supplemented male subjects (nos. 9, 14) also showed positive movement toward the mean and 2 supplemented female subjects (nos. 19, 24) showed negative movement from the mean. The change in percent median and Z—score evaluations were not always in the same direction for each subject as is shown in subject no. 17. Subject no. 26 was excluded from evaluation because she had already reached maturity. Change in chronological age and bone age for each subject is graphically represented in Figure 2. A comparison of the slopes for growth velocity and bone maturation are presented in Table 16. These calculations Show an increase in the slope in growth velocity (GV)in only 2 subjects (no. 17, 24) with zinc acetate supplementation, but an increase in bone maturation (BM) in 4 of the 6 zinc supplemented subjects (nos. 5, 17, 15, 19) for whom data were available E. Tanner Staging Tanner sexual maturity scores for male subjects per Chronological decimal age and the percent of normal are Presented in Table 17. Identical information is given for females in Table 18. All subjects remained at their same 61 level of sexual maturity regardless of zinc supplementation although the percentile of the reference population changed in some cases. Subject 5 appeared to be advanced for his age, while male subjects 9 and 14 appeared to have a delay in sexual maturity for their age. Female patients maintained their same level of sexual maturity for decimal age. A dash in Table 17 indicates that the sexual maturity rating per age fell outside the parameters of the reference data. Consequently, a percentile comparison could not be noted. F. Biochemical Measurements The normal range of blood chemistry values used at Children‘s Hospital of Michigan are presented in Table 1. Individual mean Chemistries for male and female subjects are presented in a series of Tables (19-29). As would be expected in children with ESRD, mean blood urea nitrogen (BUN) and serum creatinine concentration (Table 19) were above the normal range for each subject. However, the range of values for BUN and serum creatinine appeared to increase in males during the zinc supplemented period (period II). In females, the range for BUN seemed to decrease during the supplemented period, while serum creatinine values also appeared to rise. Mean total CO2 (Table 20), which is an indicator of acidosis, was maintained within the normal range 62 in all cases except subjects no. 15 and 26. Both patients 15 and 26 were being treated for their ESRD using HD. The low mean C02 values occurred during the zinc supplemented period. Alkaline phosphatase (Table 20) values were consistently above the normal range in most subjects both pre— and post— supplementation. The exceptions were subjects no. 18, 23, 10 in whom normal values occurred both pre— and post—zinc supplemention, and in subjects no. 14 and 26 pre- supplementation and subject no. 19 post—supplementation. Therefore, of the 12 subjects studied, 5 males and 2 females had high mean alkaline phosphatase values when not supplemented with zinc and, of the 9 zinc supplemented subjects, 3 males and 2 females had high mean alkaline phosphatase values. However, with zinc acetate supplementation, mean alkaline phosphatase values increased in 3 out of 4 male subjects and 4 out of 5 female subjects. Since alkaline phosphatase is a zinc dependent enzyme, correlation coefficients were calculated between alkaline phosphatase and plasma zinc levels (Table 21). Limited number of values were available for testing of significance. However, one male and one female subject were found to have Significant correlations between their plasma zinc values and their alkaline phosphatase levels when non-zinc supplemented. Mean serum sodium and chloride levels were maintained Within the normal range for all subjects when supplemented 63 and when non—supplemented with zinc (Table 22). Mean serum calcium and magnesium values hovered around the normal range, while mean serum phosphorus levels were high in 5 out of 7 males, 2 out of 5 females pre—supplementation and 3 out of 4 males, 4 out of 5 females post-supplementation (Tables 23 and 24). Although mean serum calcium values were in the normal range, mean serum calcium values increased in 5 out of 9 subjects and remained the same in 2 subjects when zinc supplemented. Mean serum phosphorus levels usually show an inverse relationship with calcium, but this was only true for half (4/9) of the zinc supplemented subjects. High mean serum magnesium levels were noted in subjects no. 1, 19, 26. These high magnesium levels may have been due to recent blood transfusions. High mean serum potassium values were found in 1 male and 2 female subjects before zinc supplementation, and 2 different male, but the same two female subjects after supplementation with zinc. Mean hemoglobin and hematocrit values were below the normal range for children in all cases (Table 25). Total Serum protein mean values were within the normal range for all subjects except no. 17 (Table 26). Mean albumin levels, however, were below the normal range in all cases except Subject nos. 10 and 15 post—supplementation and subject nos. 19 and 26, both pre and post—supplementation (Table 26). 64 Plasma zinc and copper and erythrocyte zinc levels are presented as mean values for male subjects (Table 27), female subjects (Table 28), and as a composite of all subjects (Table 29) both before and after zinc supplementation. Significant differences between the zinc supplemented and the non—zinc supplemented mean plasma values were not found. Comparisons of individual mean plasma zinc values and group mean values with a normal level of 120 +/— 10 mcg/dl [1] indicate that all except one male subject had low zinc levels before supplementation and all subjects had low zinc levels after zinc supplementation. The situation is similar in the female population with only two subjects, nos. 18 and 19, showing adequate mean plasma zinc levels pre-supplementation and one subject, no. 19, within the normal range post- supplementation. Erythrocyte zinc was measured in a small number of samples. Compared to a normal adult value of 45—47 mc9/dl/gm hemoglobin/ml [1], the RBC zinc was low both before and after zinc acetate supplementation. Mean plasma copper Values, however, did not change in an inverse relationship to mean Plasma zinc levels. Male subject no. 5 had an elevated mean Plasma copper level, and male subject no. 14 presented Slightly depressed mean plasma copper. Likewise, female Sllbject no. 19 had elevated mean plasma copper levels both beefore and after zinc supplementation, and subject no. 23 65 presented a mean plasma copper level slightly above the normal range of 130 — 200 mcg/dl [1]. Blood values for BUN, serum creatinine calcium, phosphorus, sodium, potassium, magnesium, hemoglobin, hematocrit, alkaline phosphatase, 002, zinc and copper were typical of children with ESRD. G. Dietary Measurements The evaluation of individual subject dietary intake data, as shown in Appendix B, demonstrates a significant change in sodium (P<0.05), and magnesium, zinc, and iron intakes (p<0.01) between the zinc supplemented and non— supplemented periods for male subject no. 5 and for sodium (p<0.01) in male subject no. 15. Female subject no. 18 showed a significant, pre— versus post—zinc supplement, increase in sodium (p<0.05), and subject no. 23 a significant decrease in calcium (p<0.05), and phosphorus (p<0.01) in the non zinc supplemented versus the zinc supplemented period. Percent medians, using HANES II reference data for age and sex, are given in Tables 30 and 31, for kilocalories, Protein, calcium, iron, sodium, and potassium for males and females, repectively. HANES II reference data rather than 1. personal communication from Dr. A.S. Prasad 66 the RDA's were used to evaluate individual mean dietary intake data because HANES II data are representative of intakes of the American population. Approximately three quarters of the subjects had dietary intakes that were 67% or greater of the reference median intake for energy and protein before zinc supplementation and 78 percent and 89 percent, respectively, after zinc supplementation. Fifty percent of the subjects had calcium and iron dietary intakes which met 67 percent the reference median intake when non—zinc supplemented, while there was no change with supplementation for calcium, but an increase to 78 percent of the subjects meeting the 67 percent guideline for iron. HANES I reference standards (Abraham et al. 1979) for the United States population were used to calculate Z—scores for kilocalories, protein, calcium, and iron for each subject. HANES I was used to evaluate dietary intakes not only because it is representative of the American population, but also because it provides standard deviations data from which Z-scores can be calculated. Z-scores for individual subjects and nutrients are presented in Table 32. Results indicate adequate dietary intakes of these four nutients in all cases. However, subject no. 5 demonstrated a negative Variation from the mean by more than 1 standard deviation for kilocalories, protein, and iron when non—zinc supplemented and protein during zinc supplementation. Likewise, subject 67 no. 10 varied from the mean by 1 standard deviation for kilocalories and calcium regardless of zinc supplementation status and male subject no. 15 had a Z-score of —1.01 for calcium when zinc supplemented. Only female subject no. 23 showed a negative standard deviation of greater than 1 for calcium while zinc supplemented. Dietary intake records show Z-scores that are less than 2 standard deviations from the mean for all subjects for kilocalories, protein, calcium, iron. A comparison of the dietary intake for each subject for energy and protein with the recommendations of Holliday et al. (1979) and Wassner (1982), for children with CRF are presented ianable 33. During the non-zinc supplemented period, fifty percent of the subjects (6 out of 12) had energy intakes meeting at least 67 percent of their respective RDA for kilocalories, while 92 percent (11 out of 12) of the study population exceeded 67 percent of their RDA for protein. The percentage of the study population meeting 67 percent of the RDA's increased to 67 percent (6 out of 9) for energy and 100 percent for protein with zinc acetate supplementation. However, subject no. 14 who had low protein intakes in the non—supplemented period was not included in the study period after zinc supplementation so protein intake was similar before and after zinc supplementation. Dietary zinc intake was at 67 percent of the RDA in 42 percent (5 out of 12) of the study 68 population before zinc supplementation and increased to 67 percent (6 out of 9) of the subjects during the zinc supplemented period. H. Growth Evaluation The onset of puberty can effect growth velocity. Decimal ages shown in Tables 8 and 9, indicate that 5 of 7 males in this study were older than 9 and therefore possibly in their adolescent growth spurt during one or both phases of the study. Subject No. 5, aged 7-9 during the study, maintained his same growth velocity regardless of zinc acetate supplementation. Subject 17, aged 9—11 during the two years of the study, increased his growth velocity from 1.6 cm/yr when non—zinc supplemented to 4.1 cm/yr while zinc acetate supplemented. The growth velocity of subject no. 10, aged 10—11 years throughout the study, decreased. However, Tanner staging and bone maturation remained at essentially the same level of maturity. Subject no. 15, aged 12—14 years throughout the study, demonstrated a decrease in growth velocity during the second year. Radiographs of the hand and wrist show an increase of 1.5 years in bone age in 0.8 chronological years during the first year before zinc supplement was given. For females, the data are similar. Four out of 5 69 subjects were greater than 7 years of age and therefore their growth velocity could possibly be affected by the pubertal growth spurt. Subjects no. 18 and 19, aged 14—15 and 14-16, respectively, during the study, maintained their growth velocity. The physical examination records revealed that subject no. 24, aged 11—13 during the study, began menstruation at the age of 12. This subject also grew 6.1 cm during the second year compared to 2.8 cm the first year of the study. Female subjects no. 23 and 26, aged respectively 5 t0 7 and 19 to 21 years, demonstrated decreased growth velocity with zinc supplementation. Bone maturation data on subject no. 26 shows a bone maturation of 17 at the intiation of the study. All subjects, except subject no. 26, were well below the mean height for their age as compared to the normal American population. The age of onset of ESRD, the duration of the time the children had been treated for ESRD calculated from the time dialysis was initiated through the end of the study period and the Z—scores for height at the end of the study are given in Table 34. The two children who have the greatest negative standard deviations from the mean for Insight (subjects no. 5 and 17) are also the two subjects who developed ESRD at a young age and had been under treatment for their renal failure for a number of years. Comparisons of bone age to the age of onset of ESRD 70 and the duration of dialysis treatment show a similar relationship. Subjects no. 10, 1, and 18 were recent entries into the dialysis program at Children‘s Hospital of Michigan. Their bone ages were 2.2, 0.2, 1.4 years, respectively, below chronological age before zinc supplementation, and 2.4, 1.0, and 0.6 years, respectively, below chronological age after zinc acetate supplementation. In contrast, subjects no. 5, 17, and 19, who were longer term patients, presented bone ages 5.2, 5.4, 6.5 years, respectively, below chronological age before zinc supplementation, and 5.2, 5.3, 5.4 years, respectively, after zinc supplementation. Table 1. 71 Normal Ranges of Blood Chemistries for Children at Children‘s Hospital of Michigan Constituent Range Blood Urea Nitrogen (BUN) <20 Serum Creatinine 0.4—1.3 Total Protein 5.1—8.6 Albumin 3.8—5.4 Globulin 1.28—3.16 Alkaline Phosphatase 60-230 Ferritin 10—300 Hemoglobin (Hb) 11.6-15.1 Hematocrit (Hct) 34.2-44.6 Cholesterol 80—180 Triglycerides 47-155_ Sodium (Na) 134—142 Potassium (K) 3.5—5.0 Chloride (Cl) 90—110 Calcium (Ca) 9.0-11.0 Phosphorous (P) 3.4-5.4 Magnesium (Mg) 1.6—2.7 Glucose 60—100 Carbon Dioxide (002) 20 0—29 0 mg/dl mmol/L 72 Table 2. Common Medications Used in Treatment of ESRD at Children's Hospital of Michigan Poly—Vi-Sol Multivitamin preparation used to replace vitamins removed by the dialysis process Folic Acid A vitamin that is necessary for blood formation which is removed in the dialysis process Dialume ' Phosphate binder designed to (Aluminum Hydroxide) decrease the blood phosphorus level Amphojel Phosphate binder designed to (Aluminum Hydroxide) decrease the blood phosphorus level Rocaltrol Active form of vitamin D used to replace this kidney function Tums Replacement calcium required to (Calcium Carbonate) maintain normal blood calcium levels and counteract bone calcium losses Phenobarbitol Medication designed to prevent seizures Aldomet Antihypertensive Apresoline Antihypertensive ~——--—-—----_------—-_--—----—------_--—-——---—--———-————---- * From Physician’s Desk Reference 73 Table 3. CHARACTERISTICS OF STUDY POPULATION Subject No. Sex Ethnic Dialysis Study Origin Method1 Disposition (1)2 M Asian CAPD No Zn—completed 6 mo. 2 M White HD Transplant 3 M White HD Transplant 4 M Black HD Transplant g3 M Black IPD/CAPD Completed study 6 M Black HD Death 7 M White IPD Moved from area 8 M Black HD Transferred (9) M White HD/CAPD No Zn—completed study 19 M White HD/CAPD Completed 5 mo. 11 M White IPD Unable to follow 12 M Black HD Withdrew 13 M Black HD Transplant (14) M White HD/CAPD No Zn-completed study 1; M Black HD Completed study 16 M White HD/CAPD Withdrew 11 M White HD Completed study 1g F White CAPD Completed 6 mo. 19 F Black HD/CAPD Completed study 20 F White CAPD Transplant 21 F Black HD Transplant 22 F White HD Transplant gg F Black IPD/CAPD/Completed study IPD 24 F White HD/CAPD Completed study 25 F White IPD Death gg F Black HD Completed study 1. HD - Hemodialysis CAPD — Continuous Peritoneal Dialysis IPD — Intermittant Peritoneal Dialysis 2. Subjects in parenthesis completed the study but were not supplemented with zinc 3. Subjects underlined in the first column completed the study 74 Table 4. Composition of Dialysate Solutions Used at Children's Hospital of Michigan* Chemical Eri-lyte 8306 Dianial PD-2 Solvent Solvent Sodium (mEq/L) 134 132 Calcium (mEq/L) 2.5 3.5 Magnesium (mEq/L) 1.5 0.5 Chloride (mEq/L) 101 96 Acetate (mEq/L) 37.0 Lactate 40 Dextrose (gm/L) 2.5 45 * Manufacturers' data 75 Table 5. Disease Characteristics of Participants Subject No. Primary Diagnosis Daily Urine Output Males, Zinc Supplemented 5 Hypoplastic dysplastic kidneys 500-700 cc 10 Henoch Schoelin Purpura < 50 cc 17 Focal Segmented Glomerulonephritis Anephric 15 Chronic Sclerosing Glomerulonephritis 0 cc Males, Non-Zinc Supplemented 1 Focal Sclerosing Nephrotic Syndrome 300—700 cc 9 Obstructive Uropathy Secondary to Anephric Posterior Urethral Valves at Birth 14 Acute Glomerulonephritis Anephric Females, Zinc Supplemented 18 Chronic Pylonephritis 1000 cc 19 Focal Segmented Glomerulonephritis 0 cc 23 Focal Segmented Glomerulonephritis < 50 cc 24 Hypoplastic Kidneys Anephric 26 Membranoproliferative Glomerulo- Anephric nephritis 76 Table 6. Ages, Heights and Weights of Subjects at Initiation of Study* Subject Decimal Height Weight NO- Age(yr8) (cm) (kg) Males, Zinc Supplemented 5 7.6 90.0 12.0 10 10.7 126.0 27.8 17 9.3 109.5 17.7 15 12 1 129.5 32.2 Males, Non-zinc Supplemented 1 5.7 100.0 16.8 9 13.6 . 131.4 32.6 14 15.5 149.0 38.1 Females, Zinc Supplemented 18 14.8 149.0 49.7 19 14.3 135.0 29.1 23 5.2 96.0 14.2 24 11.8 127.0 20.5 26 19.6 154.9 57.6 * Subjects started at various times for different time periods. 77 Table 7. Prediction of Adult Height By Various Methods for Male and Female Subjects Subj- CAa BAb HT Midparent PMHC PMHd z—scoree No. (yr) (yr) (in) Stature Garn Bayley & Rohmann' Pinneau (in) (in) (in) Males 5 9.6 4.5 40 67.5 53.9 — — 10 11.4 9.0 50 64.3 54.5 63.6 -2.35 17 11.3 6.0 45 65.0 56.7 66.2 —1.41 15 13.9 11.0 54 — — 65.6 -1.63 1 6.6 4.5 41 44.7 59.0 - - 9 14.6 10.0 53 68.5 67.0 65.3 ~2.18 14 16.6 12.0 59 66.0 69.0 69.8 -0.10 Females 18 15.6 15.0 60 65.0 64.0 60.6 -1.48 19 16.4 11.0 56 - - 61.0 -1.31 23 6.4 4.2 40 68.5 66.0 - - 24 13.7 10.0 53 66.8 62.5 60.6 -1.48 26 20.9 17.0 62 - - 62.0 -0.87 a CA-Chronological Age b BA-Bone Age c PMH-Predicted Mature Height, Garn and Rohmann, 1966 d PMH-Predicted Mature Height, Bayley and Pinneau, 1952 e Denotes predicted standard deviation from mean adult height based on Bayley and Pinneau method 78 Table 8 . Growth Velocity - Males Subject Age Non-Zinc Supplemented Zinc Supplemented No . yrs CID/Yr r s lope cm/yr r slope 5 7 5.9 0.98b 5.32 5.7 0 95b 5.17 10a 10 3.9 0.70 3.17 1.4 0 11 0.53 17 9 1.6 0.70c 2.04 4.1 0 76C 2 67 15 12 6.6 0.89b 5.55 0.7 -0 03 —0 09 1 5 5.3 0.48d 0.55 — - - 9 13 3.8 0.05 0.35 - - - 14 15 1.4 0.60d 2.24 - - - Mean 4.07 2.75 2 98 2 31 SD 2.02 2.03 2.33 1 88 * Correlation between height and decimal age a 5 months growth extrapolated to 1 yr. b Statistical significance P<0.01 c Statistical significance P<0.02 d Statistical significance P<0.05 79 Table 9. Growth Velocity — Females Subject Age Non—Zinc Supplemented Zinc Supplemented No . YI‘S cm/yr 1‘ slope cm/yr r* slope 18 14 4.5 0.92b 3.81 4.4 0.27 2.75 19 14 3.8 0.88b 3.29 2.4 -0.15 0.55 23 5 4.6 0.94b 5.12 3.0 0.59 1.91 24 11 2.8 0.50 3.88 6.1 0.70C 6.11 26 19 3.7 0.76 3.47 0.5 0.43 2.05 Mean 3.88 3.91 3.28 2.67 SD 0.73 0.72 2.10 2.07 * Correlation between height and decimal age a 6 month growth data extrapolated to 1 yr b Statistical significance P<0.01 0 Statistical significance P<0.02 d 13 months growth post—supplementation reduced to 1 year 80 Table 10. Height per Age, Weight per Agea of Male and Female Subjects Expressed as Z-scores (2) Subject Beginning Crossover End of No . of Study Point Study Ht/Age WT/Age Ht/Age Wt/Age Ht/Age Wt/Age Z Z Z Z Z Z Males 5 -6.63 -3.11 -6.68 -3.47 -6.05 -1.92 10 -2.30 —0.93 -2.60 -1.25 -2.68 -1.38 17 —4.59 -2.03 -4.41 —1.82 ’-4.56 -2.03 15 -2.64 —0.85 —3.00 -1.05 -2.39 —1.61 1‘3 — - —3.02 -1.16 -2.13 -0.85 9‘9 — - —3.25 -l.82 -4.32 -2.28 _ 14b - - -3.13 -2.04 -3.32 -2.78 Females 18 —1.74 +0.55 -2.06 +0.17 —1.84 +0.25 19 -4.11 -2.24 -4.00 -2.24 -3.08 -1.83 23 -2.43 —1.62 -2.80 —1.04 -3.40 -1.97 24 —3.11 ~2.22 -3.98 -2.19 -4.50 —2.07 25 —1.42 -O.19 -O.80 -O.81 -0.65 —O.68 a Reference standards from HANES I (Hamill et al. 1977; Hamill et al. 1973) b No zinc supplement 81 Table: .11. Correlation Coefficients for Height per Age, Weight per Age,and Weight per Height for Male and Female Subjects r values Subjeczt: Non-Zinc Supplemented Zinc Supplemented No. Ht/Age Wt/Age Wt/Ht Ht/Age Wt/Age Wt/Ht Males 5 0.98a 0.93a 0.60C 0.95a 0.95a 0.95 10 0.70b —1.00 —1.00 0.11b -0.29C -0.52 17 0.70 0.92a 0.63C 0.76 0.73 0.78 15 0.89a 0.69 0.89b -0.03 0.36 0.16 1 0.48c 0.73 0.52 — — - 9 0.05 -0.23 0.19 - — - 14 0.60C -0.37 —0.26 — - - Females 18 0.92a 0.94a 0 79c 0 27 0 39 -0.03 19 0.88a 0.23 0.60 -0.15 0.58 -0.09 23 0.94a 0.90a 0.76C 0.59 0.35 -0.41 24 0.50 0.87b 0.27 0.70b 0 77a 0.34 25 0.76 -0.88a —0.64 0.43 0 60 0.68 a Statistical Significance P<0.01 b Statistical Significance P<0.02 c Statistical Significance P<0.05 82 Table 12. Weight per Height of Male and Female Subjects Expressed as % Median* Subject Beginning Crossover End of No . of Study Point Study 5 .. - _ 10 109 108 108 17 098 104 113 15 - 108 113 1 - _. .. 9 — _. _ 18 117 132 134 19 - - - 23 - - — 24 - - — 26 - - - *Reference standards from HANES I (Hamill et al. 1973). Missing calculations indicate that individual data was outside the range of reference standards. 83 Tables 13. Arm Muscle Circumference (AMC) Expressed as Percent Median, Mid—Arm Circumference (MAC), and Triceps Fatfold (Tsfifl) Expressed as Z—scores (Z) for Male and Female Subjectsa Subject Period I Period II No. ------------------------------------------------ MAC AMC TSF MAC AMC TSF z x z z x z med. med Males 5 —1.93 96 -1.04 -0.65 106 -0.84 10 -0.70 92 +0.23 10.58 101 —0.43 17 -1.83 84 -0.73 -1.58 85 -0.81 15 -0.42 105 —0.23 —0.79 106 -0.60 1: —1.57 88 -0.39 -0 20 97 +0.71 9b -1.48 94 -0.69 —1.43 91 -0.63 14 -1.59 88 -0.53 -2.82 80 -1 33 Females 18 +1.53 126 +0.92 +1.21 109 +1.98 19 -0.92 87 —2.39 —1.33 92 —0.75 23 -0 71 106 —1.02 —1 21 98 —0.95 24 -1.67 92 —0.95 -1.42 94 -1.04 26 - 103 - - 114 - a Reference standards HANES I (Johnson et al. 1981; Johnston et al. 1974) b No zinc supplement Some .33 6:6 £03523 woe wcom no 503335 program 0 coflvmucwewfiodom oofiN mo meeccwmwm n Acumfi .Hm um whoom “vows .Hm um Maoomv H mmzcz Eonm humongoum moempMMQm m 84 mm.eI on v.0H O.NH m.oH mo.vI me O.HH m.Ha m.ma mo.NI we o.HH m.HH ©.vH ea em.el mm m.HH o.OH N.¢H Hm.MI No m.HH m.w m.MH ov.wl mm n.0H o.~ O.Na m I mm ¢.m m.v «.0 I no m.h o.v N.v I I m.w o.v N.¢ H ocfiN 02 I mmamz mm.NI mo n.0H o.HH o.¢m ov.NI No o.HH m.m «.mH mo.HI as N.HH o.m N.HH, ma mm.¢l mm m.m o.o m.HH 0H.VI me o.m o.m v.0H om.MI on 0.0 o.m H.m NH om.HI ms m.HH o.m e.HH mm.HI on m.HH m.m c.0H I I I I I OH mo.ml we v.m m.¢ N.m om.mI ov o.h m.m h.m mo.vI om m.m m.N m.o m OCHN I mmflmz muoom OE mp> mh> whoom OE mn> mn> mnoom OE mh> mn> IN cox om em <0 IN cox om ommono coucmemaddom ocfiNIooz .noom mmuomhnom mam: new Acme was wcom pew Amo cementum o cofipmuomEmHodom oceN mo mosecwmmm Q lemma .e6 e6 wroom lemma .e6 um mcoomv H maze: soup monmocmum mocmamcmm m I I I I I I I I o.~H m.oN I I I o.~H «.me 0m 0e.mI an 0.0H o.oH h.mH mm.al we m.oH o.oH m.me 0N.HI me m.m m.m o.NH em I I I I I mm.NI co m.~ N.e «.0 I I I I I mm ee.0I hm m.NH o.HH e.0H mm.ml mm m.m o.m m.mH «0.mI on 0.0 o.m o.eH ma oc.o+ 0m «.ma o.mH 0.0H 0m.OI Hm m.¢H m.ma m.vH I I I I I ma whoom OE new mu> whoom OE mn> mn> whoom OE mp> mp> IN <0x om 97 7.1 82 >97 24 12.7 Bl.P2 <3,50 13.7 B1/2,P2 - 26 20.6 B4,P5 <3 - - — a Centile reference from Buckler, 1979. Missing data indicates that readings were not taken. Incorrect scoring on this subject B=breast, P=pubic hair 00' 89 Table 19. Individual Means for BUN and Serum Creatinine for Male and Female Subjects Subj. Non—Zinc Supplemented Zinc Supplemented No BUN Creatinine BUN Creatinine mg/dl mg/dl mg/dl mg/dl N* x SD N x SD N x SD N x SD Males 5 11 43 15.4 11 7.0 0.73 12 61 12.06 11 8.9 1.27 10 5 29 3.27 5 9.5 0.59 5 47 11.57 4 8.9 0.52 17 3 67 29.5 3 9.1 1.25, 9 75 13.67 9 9.9 0.89 15 7 99 16.96 7 12.0 2.60 11 90 7.27 10 13.6 0.77 Males - No Zinc 1 10 51 13.8 10 4.7 0.53 — — — - - - 9 12 72 12.60 11 8.4 0.57 — ~ — — — — 14 12 56 5.16 12 11 5 0.69 - - - - - - Females 18 4 59 5.45 4 9.3 0.41 4 56 9.46 4 9 8 0.67 19 9 64 9.88 8 14.4 1.88 12 43 11.14 12 10.5 3.50 23 12 75 15.14 12 8.6 0.69 9 68 18.13 9 8.9 1.12 24 11 67 13.67 10 10.7 1.74 12 58 8.97 12 11 2 1.11 26 12 66 9.65 9 14.5 0.84 10 83 12.45 9 15 0 1.02 * N-number of observations; X-mean; SD—standard deviation 90 2.82 2.22 2.57 2.02 2.71 2.35 1.59 2.97 and Alkaline for male and Female Subjects 132 18 337 171 Table 20. Individual Means for Serum CO Phosphatase (Alk Phos) Subj Non-Zinc Supplemented N0 CO Alk Phos 002 mmoI/L IU/L mmol/L N* SD N x SD N x Males 5 11 23.1 2.16 0 283 95 12 27.2 10 5 26.2 2.22 5 86 17 4 24.5 17 3 28.6 1.64 2 689 276 7 26.5 15 7 22.5 8.07 6 294 95 10 18.3 Males - No Zinc 1 11 24.3 2.85 10 255 41 - - 9 11 28.9 2.04 11 342 149 - - 14 12 26.6 1.71 12 151 47 - - Females 18 4 24.0 1.46 4 95 30 4 22.5 19 8 24.3 3.13 8 284 73 10 23.5 23 11 22.0 2.43 12 130 44 7 25.0 24 7 27.0 4.83 9 635 149 10 25.1 26 1 20.9 12 210 36 7 17.6 2.76 Alk Phos IU/L N X 11 452 3 114 9 686 11 605 4 180 10 197 7 141 12 896 10 417 * N-number of observations; X—mean; SD-standard deviation 91 Table 21. Comparison of Alkaline Phosphatase (A/P) and Plasma Zinc Levels for Individual Male and Female Subjects Subject Non—Zinc Supplemented Zinc Supplemented No. N A/P Pl Zn r* N A/p p1 Zn r IU/L mcg/dl IU/L mcg/dl Males 5 - 283 127 — 6 452 111 —0.32 10 3 86 91 —1.00 - 114 - - 17 1 689 93 - 3 686 71 0.38 15 2 294 113 -1.00 5 605 108 0.09 Males - No Zinc 1 4 255 83 -0.94a - - - - 9 5 342 89 0.71 - - - - 14 8 151 98 -0.33 - — - - Females 18 3 95 126 0.92b 3 130 105 —o 43 19 1 284 112 — 4 197 151 -0 42 23 1 130 78 - 5 141 86 0.26 24 2 635 88 1.00 5 896 86 0 32 26 1 210 83 - 7 397 94 0.30 * r-correlation coefficients a Statistical significance P<0.01 b Statistical significance P<0.05 92 Table 22. Individual Means for Serum Sodium and Serum Chloride for Male and Female Subjects Subj. Non—Zinc Supplemented Zinc Supplemented No Sodium Chloride Sodium Chloride mEq/L mEq/L mEq/L mEq/L N’“ X s0 N x SD N x SD N x SD Males 5 11 138 2.21 11 101 3.14 12 135 2.43 12 95 3.23 10 5 143 2.41 5 95 4.04 5 133 3.65 5 93 5.40 17 3 136 2.52 3 97 3.21 9 137 1 87 9 98 2.49 15 7 135 4 90 7 100 3.64 10 136 1.45 11 99 1.76 Males — No Zinc 1 11 132 1.86 11 96 2.60 — — — — — — 9 11 137 2.36 11 96 2.37 - - — - - — 14 12 139 2.39 12 100 1.86 - - — - — — Females 18 4 140 1.71 4 104 2 00 4 141 1.50 4 105 2.06 19 9 135 2.99 9 97 4 56 12 133 2.70 12 95 3.70 23 12 137 2.81 12 100 2.64 9 138 3.39 9 99 3.35 24 8 138 2.00 8 99 3 25 12 137 1.88 12 98 2.97 26 9 137 2.86 9 105 2 98 9 137 1.92 9 102 1.79 * N—number of observations; X—mean; SD— standard deviation 93 Table 23. Individual Means for Serum Calcium and Serum Phosphorus for Male and Female Subjects Subj. Non-Zinc Supplemented Zinc Supplemented No. Calcium Phosphorus Calcium Phosphorus mg/dl mg/dl mg/dl mg/dl N* x SD N x 80 N x SD N x SD Males 5 11 9.9 0.80 11 5.3 1.88 12 10.3 0.62 12 3.6 1.36 10 5 10.2 0.40 5 6.0 0.95 4 10.8 0.45 4 7.3 2.72 17 2 8.2 0.42 2 7.0 0.99 . 9 8.7 0.43 9 5.5 1.56 15 7 9.8 0.46 7 7.1 2.15 11 9.8 0.40 11 8.5 1.67 Males - No Zinc 1 11 11.2 1.49 11 3.9 1.06 — — - - - - 9 12 9.5 0.39 12 6.0 1.09 - — - - - - 14 12 9.6 0.22 12 5.8 1.26 - - - — - - Females 18 4 10 8 0.46 4 3.7 0 40 4 10 0 0.24 4 4 2 1.0 19 9 9.4 0.80 9 7.1 2.27 11 10.2 0.41 10 5.8 1.59 23 12 9.4 0.30 12 7.1 1.42 9 9.5 0.85 8 5.9 1.39 24 11 9 3 0.53 11 4.9 0 96 12 9 3 0.46 12 6 2 1.65 26 11 8 6 0.41 10 4.6 1 14 9 7 9 0.96 9 5 4 1.01 * N-number of observations; X-mean; SD-standard deviation 94 Table 24. Individual Means for Serum Potassium and Serum Magnesium for Male and Female Subjects Subj. Non—Zinc Supplemented Zinc Supplemented No. Potassium Magnesium Potassium Magnesium mEq/L mg/dl mEq/L mg/dl N* X SD N X SD N X SD N X SD Males 5 11 4.0 0.46 11 2.0 0.26 12 4.1 0.41 11 2.8 0.88 10 5 4.5 0.48 5 1.8 0.22 5 4.5 0.38 4 2.9 2.32 17 3 4.5 0.90 2 1.8 0.42 10 5.3 0.59 8 1.9 0.15 15 7 4.6 0.71 — — — 11 5.3 0.43 8 2.0 0.39 Males — No Zinc 1 11 3.6 0.23 9 3.4 1 53 — — — — — — 9 11 5.5 0.31 12 1.9 0.17 — — — — — — 14 12 4.2 0.44 11 2 3 0 22 — — - - — — Females 18 4 4.1 0.42 4 2.1 0.29 4 3.6 0.18 4 2.0 0.29 19 9 4.3 0.94 6 3.5 0.51 12 3.7 0.81 6 2.7 0.31 23 12 4.5 0.56 11 3.0 0.29 9 4.6 0.59 .7 2.2 0.23 24 11 5.9 0.49 6 2.2 0.39 12 5.5 0.74 12 2.2 0.33 26 12 5.1 0.52 1 3.7 — 10 5.4 0.44 — - — * N-number of observations; X-mean; SD-standard deviation 95 Table 25. Individual Means for Hemoglobin and Hematocrit for Male and Female Subjects Subj. Non-Zinc Supplemented Zinc Supplemented No. Hemoglobin Hematocrit Hemoglobin Hematocrit gm/dl gm/dl % N*XSDNXSDNXSDNXSD Males 5 12 8.43 0.57 11 25.8 1.04 11 7.37 0.47 11 22.6 1.40 10 5 9.50 0.91 5 28.4 3.00 5 8.90 1.22 5 26.4 3.53 17 2 6.80 0.42 2 21.1 1.70 9 7.23 1.03 9 21.7 2.44 15 3 7.17 1.17 7 20.8 4.29 7 7.07 1.51 12 22.5 3.91 Males - No Zinc 1 10 9.93 1.32 10 29.7 3 93 - — - - - - 9 12 7.40 0.96 12 20.9 4 89 - - - - - — 14 12 6.94 1.63 12 20.9 4 99 - — - - - - Females 18 4 10.6 0.84 4 31.1 2 57 4 10.5 0.85 4 31.4 2.11 19 8 7.28 1.85 8 22.6 6 84 13 7.02 1.31 13 21.0 3.93 23 11 7.26 1.62 11 22.3 5.73 9 6.73 0.67 9 20.7 1.98 24 9 7.00 1.44 10 21.4 4 55 12 6.40 1.14 12 19.9 3.54 26 10 7.70 1.11 12 23.2 3 54 12 7.60 1.25 12 23.1 4.03 * N-number of observations; X-mean; SD-standard deviation 9 6 Table 26. Individual Means for Total Serum Protein and Serum Albumin for Male and Female Subjects Subj. Non-Zinc Supplemented Zinc Supplemented No. Total Protein Albumin Total Protein Albumin gm/dl gm/dl gm/dl gm/dl N* x SD N x SD N X s0 N x SD Males 5 12 5.57 0.40 12 3.29 0.39 12 5.65 0.37 12 3.18 0.41 10 4 6.13 0.17 4 3.55 0.38 4 6.73 0.17 4 4.00 0.16 17 2 4.20 0.28 2 2.33 0.16 8 4.45 0.51 8 2.52 0.36 15 — - - - - — 6 7.10 0.32 6 4.59 0.23 Males - No Zinc 1 11 5.26 0.36 11 2.53 0.19 - — - - — — 9 12 5.36 0.30 12 3.07 0.39 — — - - — - 14 12 5.29 0.37 12 3.47 0.43 - - - - - - Females 18 4 6.28 0.22 4 3.59 0.33 4 6.00 0.36 4 3.64 0.35 19 7 6.54 0.90 7 4.16 0.72 12 6.65 0.65 11 3.93 0.61 23 12 5.31 0.47 12 3.10 0.60 7 5.23 0.28 7 2.72 0.31 24 7 5.39 0.56 7 3.39 0.67 11 5.85 0.34 11 3.50 0.77 26 5 6.14 0.27 6 4.07 0.20 7 6.66 0.38 8 4.20 0.26 * N—number of observation; X—mean; SD—standard deviation 97 Table 27. Individual Mean Plasma and RBC Zinc and Plasma Copper Levels for Male Subjects Subj Non—Zinc Supplemented Zinc Supplemented No Zinc Copper Zinc Copper Plasma RBC Plasma Plasma RBC Plasma mcg/dl mcg/dl mcg/dl mcg/dl mcg/dl mcg/dl 5 127(3)* 44(2) 218(3) 111(6) ' 33(5) 153(8) 10 91(3) - 189(3) 107(3) 40(1) 200(3) 17 93(2) - 133(2) 71(5) 42(4) 137(5) 15 113(3) 52(1) 192(2) 108(6) 40(4) 173(6) 1 83(4) 32(2) 175(4) - - - 9 89(5) 34(2) 163(5) - - — 14 98(8) 44(4) 127(8) - - - * Number of samples 98 Table 28. Individual Mean Plasma and RBC Zinc and Plasma Copper Levels for Female Subjects Subj Non-Zinc Supplemented Zinc Supplemented No Zinc Copper Zinc Copper Plasma RBC Plasma Plasma RBC Plasma mcg/dl mcg/dl mcg/dl mcg/dl mcg/dl mcg/dl 18 126(3)* — 192(3) 105(3) 41(2) 188(3) 19 112(1) — 225(1) 151(6) 49(2) 226(6) 23 78(1) - 142(1) 86(7) 34(3) 202(7) '24 88(2) 27(1) 169(2) 86(5) 36(2) 172(5) 26 83(1) - 133(1) 94(7) 39(4) 129(7) * number of samples Table 29. Male and Female Group Mean Zinc and Copper Blood Levels for and Males and Females Combined Blood Non—Zinc Supplemented Zinc Supplemented Component No.3 mean standard dev. mean standard dev. Males (7)b plasma Zinc 34 98.6 17.6 99.2 26. RBC Zinc 14 40.6 6.8 38.5 5. plasma Copper 33 160.8 42.5 161.2 45. Females (5) plasma Zinc 9 91.3 14.1 103.9 33. RBC Zinc 1 27.4 0 38.9 6. plasma Copper 9 176.4 42.2 182.0 44. Males and Females (12) plasma Zinc 43 97.1 17.1 101.9 30 RBC Zinc 15 39.9 7.4 38.8 5. plasma Copper 42 164.2 42.4 172.8 45. a Number of samples before zinc supplementation includes more than 1 years data b Number of subjects in parenthesis includes both zinc supplemented and non—supplemented 100 Table 30. Individual Mean Dietary Intakes for Males Evaluated as Percent Median of HANES II* Subject No of % Median No. Food ------------------------------------------- Diaries Kcal Pro Ca P Fe Na K 5 13 67 61 58 76 36 79 38 10 5 62 76 62 65 57 51 88 17 11 76 82 96 79 80 99 81 15 2 68 108 51 68 83 57 74 1 7 53 49 57 49 49 44 51 9 9 83 94 148 112 53 82‘ 118 14 9 50 41 43 35 45 58 41 Zinc Supplemented 5 10 69 53 53 51 60 40 35 10 4 74 93 42 73 97 58 88 17 8 85 96 97 85 96 103 81 15 2 63 71 42 51 67 40 75 * Reference standards from HANES II (Carroll et al. 1983) 101 Table 31. Individual Means for Dietary Intakes for Females Evaluated as Percent Median of HANES II* Subject No. of 2 Median No. Food Diaries Kcal Pro Ca P Fe Na K ‘Non-Zinc Supplemented 18 4 94 113 144 116 124 83 137 19 9 115 117 74 100 132 124 126 23 12 68 92 53 79 64 63 80 24 7 71 75 80 76 83 70 72 26 5 86 98 92 83 99 74 102 Zinc Supplemented 18 4 118 102 127 109 124 165 83 19 8 133 148 123 126 210 173 138 23 4 63 77 27 33 60 59 52 24 8 85 82 75 77 83 81 86 26 5 86 98 92 83 99 74 102 * Reference standards from HANES II (Carroll et al. 1983) 102 Table 32. Dietary Intakes for Kilocalories (Kcal), Protein (Pro), Calcium (Ca), Iron (Fe) for Male and Female Subjects Expressed as Z-scores (Z) Subj. NO- 0f* Non—Zinc Supplemented Zinc Supplemented No Food Diaries Kcal Pro Ca Fe Kcal Pro Ca Fe Z Z Z Z Z Z Z Males 5 13/10 —1.02 —1.13 ~0.67 —1.74 -0.96 —1.25 -0.77 -0.81 10 5/4 -1.34 -0.91 —1.01 -0.80 -1.01 —0.50 -1.36 -0.00 17 11/8 -0.90 -0.75 -0.41 -0.35 -0.69 -0.47 -0.38 —0.02 15 2/2 -0.84 -0.33 -0.81 -0.18 -0.98 -0.61 -1.01 -0.40 Females 18 4/4 —0.31 +0.10 +0.11 +0.48 +0.26 -0.14 -0.09 +0.47 19 9/8 +0.09 +0.08 —0.53 +0.43 +0.44 +0.59 +0.18 +1.72 23 12/4 -0.76 -0.28 -0.79 -0.67 —O.89 -0.64 —1.51 -0.76 24 7/8 —0.93 —0.88 —0.60 —0.46 —0.59 -0.75 -0.66 —0.47 26 5/5 —0.24 -0.16 -0.04 -0.11 +0.40 +0.28 +0.05 +0.32 * Slash separates non—zinc supplemented and zinc supplemented number of food diaries 103 Table 33. Recommended Nutrient Levels and Percentage of Recommended for Male and Female Subjects SUPJECt Recommendeda % Recommended 8 Recommended No. 1 2 non-supplemented Zn—supplemented Kcal Pro Pro Zn Kcal Pro Zn Kcal Pro Zn gm gm mg % gm% mg% % gm% mg% 5 1300 24 29 10 98 175 49 102 134 124 10 2400 56 55 10 54 100 50 64 125 94 17 1700 35 40 10 95 174 71 104 175 73 15 2400 64 68 10 66 140 121 61 87 73 1b 1700 34 10 48 79 44 9b 2400 67 10 80 116 90 14b 2700 76 15 49 52 27 18c 2200 50 60 15 70 136 68 88 102 51 19 2400 58 62 10 78 121 77 90 144 116 23 1300 28 34 10 92 200 63 85 134 55 24 2400 41 54 10 53 91 59 63 91 69 26c 2200 58 53 15 62 100 50 86 136 64 a Reference for Kcal from Recommended Dietary Allowances, National Research Council (RDA,NRC); protein calculated at 2 gm protein per kg body weight (Holliday et a1. 1979), 1) protein calculation based on weight at initiation of study, 2) protein calculation based on weight at beginning of supplemented period b No zinc supplement c Protein calculated at adult recommendation of 1 gm protein per kg body weight (Holliday et al. 1979) 104 Table 34. Relationship of Z-scores for Height, Age of Onset of ESRD, and Duration of Dialysis for Males and Females Subject Age of Onset Duration of Height No. ESRD(yrs) Dialysis(yrs) Z—score Males 5 birth 7.9 -6.05 10 11 0.8 -2.68 17 5 5.9 —4.56 15 8 6.3 -2.39 1 6 0.7 -2.13 9 11 4.3 -4f32 14 10 7.7 -3.32 Females 18 15 0.7 -1.84 19 10 5.9 -3.08 23 3 4.1 -3.40 24 10 4.6 -4.50 26 8 14.6 —0.65 Figure 1: Growth Velocity of Subjects With and Without Zinc Acetate Supplementation .H madman ¢<=_ . fl ¢_ I — v ”:54: 5" cm 8:: III \ ‘ \ l \‘\\ \ l“‘ I ma V 5““ I E: . r|||| \ NR we \ a: n.:\ u: N 2:! 4] IV 02. .lI a: N—zaZZB I. -“‘ll III-III ““ k 5.. I... $ \ 3:9‘ .__ we ‘Iun 92“ ||nv|| “ ||||.I“ Izl|| o2 ee_.|||l II‘|‘I :3 u: W3 NI lHUHH Figure 2: Mean Skeletal Age at Each Chronological Age (Diagonal Line) From Greulich and Pyle. Radiographic Atlas of Skeletal Development of the Hand and Wrist, 1959. Subjects Before ZN — *. Crossover Point — 0, After ZN - [] .N gunman safe a: 2285285 2 E E o a. a N as N moss: I I .I \* was. I \ W . X E e \. t w‘ \rr. .1 \ . 0. a» me ~ \ . 0.10 a c N— D N E i L e (SHVSA)39V1V1313XS V. DISCUSSION OF RESULTS A. Introduction Growth retardation in children with CRF was evident in this study as has been reported by other investigators (West and Smith, 1956; Potter and Greifer, 1978; Boyer et al. 1974; Kleinknecht et al. 1980; Salusky et al. 1983). This investigation also demonstrated that this growth failure is associated with decreased growth velocity, decreased bone maturation per chronological age, decreased sexual maturation for age, decreased AMC and TSF measurements for age and sex, increased BUN, serum creatinine, and alkaline phosphatase values, low hemoglobin, hematocrit, albumin, and plasma zinc levels, and in some cases inadequate nutrient intake. As in other studies (Boyer et al. 1974; Betts and Magrath, 1977; Simmons et al. 1971; Balfe et al. 1981; Salusky et al. 1982), population numbers are low and the characteristics of the population varied. Seven males and 5 females completed this study. Since this was a longitudinal stmiy encompassing two years, it was difficult to keep subjects for the entire time. Subjects were lost to the study primarily due to the normal course of CRF and prudent medical care (Table 1). At the initiation of the study, 4 of the 7 males were prepubertal the other 3 either in or 108 entering the pubertal phase (Tanner and Whitehouse, 1976) of development (Table 8). Likewise, three females were entering or in the pubertal growth phase, one beyond this physical developmental period and one prepubertal (Table 9). Salusky et al. (1983) split their study population into children less than 10 years of age and children greater than 10 years to account for the effect of puberty on growth. However, the population characteristics in this study limited such global evaluation of data. Therefore, in addition to discussing the results collectively, data on each subject is presented separately in Appendix B as individual cases. With all clinical studies, the investigator is dependent upon the cooperation of the study population, in this case. Children, to obtain reliable data from which conclusions can bacirawn. In a long-term study, such as the two year time Pericnd in this study, the risks of facility, personnel and environmental changes, which affect the research design, are increased. Likewise, in a population where dysgeusia, anorexia, social and psycological problems, and medical complications are common, the risks of non compliance and leek of cooperation are increased. Also, in clinical investigations, the cooperation of many health professionals is required for the collection and processing of information. Inherent in this process is the possiblity of error. 109 B. Growth Measurements Stefanidis et al. (1983) reported improved growth velocity in children treated with CAPD compared to those treated with HD and IPD. Salusky et al. (1983) found a significant positive correlation between TSF and duration on CAPD. Brem and Toscano (1984), however, conclude that no form of dialysis treatment holds any advantage over another in terms of growth in children with CRF. In our study Population, 1 male and 1 female were treated with HD and the remaining 10 subjects were treated using CAPD. One patient Mes treated with CAPD and periodic HD due to a decrease in ultra-filtration of the peritoneum. Due to the small numbers amiages of the two patients in our study who were treated With HD, it is impossible to draw conclusions from our data Withe effect of treatment method on growth velocity, and bmm maturation. A number of authors (Betts and Magrath, 1974; Mehls et a1.1978; Boyer et al. 1974; Scharer and Gilli, 1984) note that there appears to be a relationship between the extent of the 9rOWth retardation, the age of onset ESRD, and the duration of dialysis. This is evident in this study also. TWO children who have the greatest negative standard deviations from the mean for height (Table 34) are also the 110 two subjects who developed ESRD at a young age and have been under treatment for their renal failure for a number of years. In addition to statural growth retardation, Kleinknecht et al. (1980) determined that the level of bone maturity was retarded for chronological age and that this increased the time span for statural growth to occur. This is evident in subject no. 26 who continued to grow through the age of 20. Roche and Davila (1976) evaluated the reliability of assessments of maturity of hand wrist radiographs using the Gruelich and Pyle atlas.. They concluded that bones of the hand—wrist should be assessed in a random order, that a Single age should be given to groups of bones and that carpals should be excluded from the assessment. Hand-wrist radiographs for subjects in this study were read in this manner. Anthropometric measurements provide a rough estimate of bOdY composition. Arm muscle circumference estimates the amount of muscle and TSF is an estimate of body fat (Frisancho, 1974). Therefore, according to the percent median for AMC (Table 13) there appears to be a trend toward an increase in muscle in the zinc supplemented group. Salusky et al. (1983) reported a significant direct correlation between Z-scores for TSF and months of treatment With CAPD and a weak correlation between Z-scores for MAC and 111 duration of CAPD. These authors suggest that this improvement with CAPD treatment "could be due to the glucose absorbed from the dialysate and, possibly, the periodic surges of hyperinsulinemia which occur after each exchange with hypertonic glucose". In our study (Table 13), 4 out of 8 subjects who demonstrated improved Z-scores for TSF, and the 4 out of 8 subjects who showed improved Z-scores for MAC, were all treated with CAPD and received zinc acetate supplementation. However, 2 of the 3 subjects followed who did not receive zinc supplementation but were treated with CAPD, also had improved Z-scores for MAC and TSF. Although Salusky et al. (1983) did not report an improvement in AMC with CAPD treatment, our data show an improved percent median for AMC in 5 of 7 subjects treated with CAPD and zinc supplement (71%), and 1 of 3 subjects treated with CAPD Without zinc supplement (33%). This suggests that zinc may have a positive effect on muscle mass and may enhance the POSitive effect of CAPD therapy on TSF and AMC. However, CAPD appears to be more beneficial than does zinc supple— mentation on improvement in TSF and AMC. Increased energy and protein intake could also be responsible for the improvement seen in MAC, AMC, TSF (Tables 13 and 32). In 2 of 4 subjects with improved Z-scores for MAC and TSF after zinc supplementation, there was also an improvement in the energy intake. The kilocalorie Z—score in 112 these 2 subjects (nos. 17 and 24) increased from —0.90 and - 0.93 to -O.69 and -O.59, respectively, with zinc acetate supplementation. Both of these subjects were on CAPD, were beginning puberty during the second year of the study, and were also the 2 subjects that demonstrated increased growth velocity with zinc supplementation. In 5 out of 7 subjects who demonstrated improved percent median for AMC with zinc acetate supplementation, the energy intake was also improved and 3 were being treated with CAPD and 2 by HD. The protein intake Z-scores were also improved in 5 of the 7 subjects with improved AMC percent median during zinc acetate supplementation. All.these 5 subjects were on CAPD therapy. Therefore, it would appear that adequate energy intake may have spared protein as an energy source in some subjects, thus allowing for increased muscle mass. Adequate zinc may have also been beneficial to this anabolic process. It is also known that DNA and RNA polymerases, required for protein biosynthesis, are zinc dependent enzymes (Coleman, 1983). Consistent with the observation of increased protein intake and metabolism and the improved AMC after zinc acetate supplementation was the increase in mean BUN in 4 of 7, and serum creatinine in 5 of 7 subjects. However, it is clear that the improvement seen in MAC, AMC, and TSF, is related to many factors. Zinc may be only one of these factors. USing Bayley and Pinneau's (1952) method (Table 7), one 113 can predict that under optimum conditions, this child population will be 1 to 2 standard deviations less than the mean adult height in all cases except subjects no. 14 and 26. These predictions are based on normal growth rates occurring at the time of the height observation and calculation. The Bayley and Pinneau, (1952) method was used because it is geared to the reading of radiographs using the Greulich and Pyle Atlas. Other methods presented in Table 7, give conflicting predictions. However, all methods were developed to evaluate normal children, not children compromised with a chronic disease condition. Therefore, thesaapredictions are of limited value. Height per age Z- SCOJres present an even more dismal prediction of adult Statrure. Z-scores for height per age range from a low of ‘2-1.3 standard deviations to a high of -6.05 standard deViations from the mean for males, and -0.65 to -4.50 Starndard deviations from the mean for females. These pre0.01) Figure 21: Hand Wrist Radiograph of Subject 23 Before Zinc Supplementation: Chronological Age 6.4 Years; Bone Age 4.2 Years 177 178 Subject No: 24 Description: White, Female Primary Diagnosis: Hypoplastic Kidneys Age of Onset: 10 years Duration of Dialysis Therapy: 4.6 years Dialysis Treatment: CAPD & HD; lOOOcc 5 times/da; PD—2 — 2.5% Medications: Multivitamin; Folic Acid; Inderol; Aldomet; Apresoline; Alternajel; Rocaltrol; Calcium Carbonate. Non Zinc Supplemented Zinc Supplemented Decimal Age: 11.8 yrs 12.7 yrs Bone Age: 10.0 yrs 10.0 yrs Tanner Score: Bl,P2 B1~2,P2 Anthropometrics: MAC: 5th 5th TSF: 5th < 5th Chemistries* -Zn +Zn Dietary Intakes* —Zn +Zn BUN: 67 58 Kcal: 1260 1505 Creatinine (mg/d1):1o.7 11.2 Protein (gm): 45 49 Na (mEq/L): 138 137 Ca (mg): 633 594 K (mEq/L): 5.9 5.5 P (mg): 809 821 Cl (mEq/L): 99 98 Na (mg): 1617 1874 Ca (mg/d1): 9.3 9.3 K (mg): 1440 1726 P (mg/d1): 4.9 6.2 Mg (mg): 125 147 Mg (mg/d1): 2.2 2.2 Cu (ug): 369 648 CO (mmoles/L): 27.0 25.1 Zn (mg): 5.92 6.85 A1 . Phos. (IU/L): 635 896 Fe (mg): 8.23 8.18 Hb. (gm/d1): 7.0 6.4 Hot. (%): 21.4 19.9 Total Pro. (gm/d1): 5.4 5.6 Albumin (gm/d1): 3.4 3.5 Zn (ug/dl): 88 86 Cu (ug/dl): 169 172 Comments: During the second phase of the study, this subject developed medical problems with CAPD. Therefore, she was dialysed using CAPD daily and HD as needed. Radiographs on this patient show severe hyperparathyroidism (arrows to phylanges). Note also the arrow to the wrist showing the development of the pisiform which occurs at 10 years in girls. * mean values Subject No: 24 Description: Age of Onset: White, Primary Diagnosis: 10 years Duration of Dialysis Therapy: 178 Female Hypoplastic Kidneys 4.6 years Dialysis Treatment: CAPD & HD; 1000cc 5 times/da; PD—2 — 2.5% Medications: Multivitamin; Folic Acid; Inderol; Aldomet; Apresoline; Alternajel; Rocaltrol; Calcium Carbonate. Non Zinc Supplemented Zinc Supplemented Decimal Age: 11.8 yrs 12.7 yrs Bone Age: 10.0 yrs 10.0 yrs Tanner Score: Bl,P2 Bl~2,P2 Anthropometrics: MAC: 5th 5th TSF: 5th < 5th Chemistries* —Zn +Zn Dietary Intakes* —Zn +Zn BUN: 67 58 Kcal: 1260 1505 Creatinine (mg/d1):10.7 11.2 Protein (gm): 45 49 Na (mEq/L): 138 137 Ca (mg): 633 594 K (mEq/L): 5.9 5.5 P (mg): 809 821 Cl (mEq/L): 99 98 Na (mg): 1617 1874 Ca (mg/d1): 9.3 9.3 K (mg): 1440 1726 P (mg/d1): 4.9 6.2 Mg (mg) 125 147 Mg (mg/d1): 2.2 2.2 Cu (ug): 369 648 CO (mmoles/LM 27.0 25.1 Zn (mg): 5.92 6.85 Al . Phos. (IU/L): 635 896 Fe (mg): 8.23 8.18 Hb. (gm/d1): 7.0 6.4 Hot. (%): 21.4 19.9 Total Pro. (gm/d1): 5.4 5.6 Albumin (gm/d1): 3.4 3.5 Zn (ug/dl): 88 86 Cu (ug/dl): 169 172 Comments: During the second phase of the study, this subject developed medical problems with CAPD. dialysed using CAPD daily and HD as needed. this patient show severe hyperparathyroidism Note also the arrow to the wrist showing the phylanges). Therefore, she was Radiographs on (arrows to development of the pisiform which occurs at 10 years in girls. * mean values Figure 22: Hand Wrist Radiograph of Subject 24 Before Zinc Supplementation: Chronological Age 12.8 Years; Bone Age 10.0 Years 179 Figure 23: Hand Wrist Radiograph of Subject 24 After Zinc Supplementation: Chronological Age 13.7 Years; Bone Age 10.0 Years 180 Subject No: 26 Description: Primary Diagnosis: 8 years Duration of Dialysis Therapy: Dialysis Treatment: Multivitamin; Calcium Carbonate; Age of Onset: Medications: 181 Black, Female Membranoproliferative Glomerulonephritis 14.6 years HD. Folic Acid; Roclatrol; Digoxin. Minipress; Non Zinc Supplemented Zinc Supplemented Decimal Age: 19.6 yrs 20.5 Bone Age: 17.0 yrs — Tanner Score: B4,P5 - Anthropometrics: MAC: 15th 50th TSF: < 5th 5th Chemistrie8* -Zn +Zn Dietary Intakes* -Zn +Zn BUN: 66 83 Kcal: 1364 1901 Creatinine (mg/d1):14.5 15.0 Protein (gm): 58 72 Na (mEq/L): 137 137 Ca (mg): 521 558 K (mEq/L): 5.1 5.4 P (mg): 798 954 C1 (mEq/L): 105 102 Na (mg): 1609 1791 Ca (mg/d1): 8.6 7.9 K (mg): 1892 2157 P (mg/d1): 4.6 5.4 Mg (mg): 139 155 Mg (mg/d1): 3 7 - Cu (ug) 683 787 (mmoles/L): 20.0 17.6 Zn (m g): 7.53 9.56 A1E.Phos. (IU/L): 210 417 Fe (m g) 9.58 11.87 Hb. (gm/d1): 7.7 7.6 Hot. (%): 23.2 23.1 Total Pro. (gm/d1): 6.1 6.7 Albumin (gm/d1): 4.1 4.2 Zn (ug/dl): 83 94 Cu (ug/dl): 133 129 Comments: The first radiograph of the wrist of this patient showed a bone age of 17 years (see following x-ray). Therefore, taken. * mean values radiographs at the end of the study were not Figure 24: Hand Wrist Radiograph of Subject 26 Before Zinc Supplementation: Chronological Age 20.9 Years; Bone Age 17.0 Years (383’