" '—-'--———-———— _— - ‘. ' ' '. . ..,. '-. 5 .. , _ . "J .. .. -:.v ..‘._ ... ' . ~. ‘. - . .. _ - ... . . . A STUDY OF THE MAGNESIUM CONTENT OE URINE AND BLOOD SERUM OF WOMEN IN RELATION TO BODY WEIGHT Thai: for tho Door» OI M. .8. MICHIGAN STATE COLLEGE Janet Sau Kin Tam 195.3. This is to certify that the thesis entitled 3 ‘. A STUDY ON THE. 1.3313531 U}: C‘QNTJII‘JT OF UTLII‘IE AIJD BLOOD SelliU‘é OF ’.JC‘.’.:JN IN RELATION TO BODY ".‘x".:JI."rHT presented by has been accepted towards fulfillment of the requirements for M degree in Mn ,’ p Major professor ‘ Date 274‘? ‘2; /9j3 D. I 0'.“ o 1-1 A DIODE UH Tun annLDlUm CUuTan OF UnINE AnD BLOOD SERUM OE women Id hELATIOh TO bODY HiIGHT By Janet Sau Kin Tam H A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of mAbTEh OF SCIENCE Department of Foods and Nutrition 1953 \ ‘ _ ill/1J3} ACKNOWLEDGMENTS The writer wishes to express her sincere gratitude to Dr. Wilma Brewer under whose patient guidance, con- tinuous encouragement and valuable assistance this study was undertaken. Grateful acknowledgment is also due to the women who served as subjects and whose cooperation made this study possible. In addition the writer deeply appreciates the graduate assistantship provided by the Foods and Nutrition Depart- ment of Michigan State College which helped to make this new experience accessible. The author is indebted to Betty Hawthorne, Eva Hwang and Chuan Huan Wu for their helpful advice and generous cooperation throughout this study. I" N 1 . Lid‘t’i .i. l TAb L11. Cir“ C \J n 'i‘ri'. TS CHAPTER INTRODUCTION . . . . . . . . . . . . . . . . . HEVIEIJ 0P1 LI‘iF-HIJIIURE o o o o o o o o o o o o 0 Distribution of Magnesium in the Body . . . Factors Affectinr the Magnesium Content of Blood Factors Affectin? Macnesium Ketabolism . . . Distribution of Madnesium in Foods . . . . . Chemical Methods . . . . . . . . . . . . . . EXPhnIKEETAL PROCEDURE , , . , , . . . . . . . EXperimental Plan . . . . . . . . . . . . . Magnesium Content of Blood Serum . . . . . . Magnesium Content of Urine . . . . . . . . . RESULES AuD DISCUBSIUn . . . . . . . . . . . . Variations in Serum and Urinary Magnesium in Individuals . . . . . . . . . . . . . . . Serum and Urinary Magnesium in Relation to Body Weight . . . . . . . . . . . . . . . Physical description of subjects . . . . Serum magnesium . . . . . . . . . . . . Urinary mannesium . . . . . . . . . . . Relationship of dietary magnesium to serum and urinary marnesium . . . . . . . . SUMMAHY'AKD CONCLUbIJLS REEEhELCES CITED 11 16 18 21 23 26 29 29 LIST OF TABLES TABLE I. A Compilation of Blood Kapnesium Values Reported for Healthy Individuals II. The Recovery of Added Magnesium from Blood Serum III. The Recovery of Added Magnesium from Urine IV. A Comparison of Venous and Capillary Magnesium V. Diurnal Rhythm of Serum Nannesium VI. Variations in Serum, Urinary and Dietary Magnesium for Seven Subjects VII. Physical Description of Overweight Subjects VIII. Physical Description of Average Weight Subjects IX. Physical Description of Underweight Subjects X. The Magnesium Content of Urine and Serum of Overweight Women and the Dietary Intake of Magnesium XI. The Magnesium Content of Urine aid Serum of Average Weight Women and the Dietary Intake of Magnesium XII. The Magnesium Content of Urine and Serum of Underweight Women and the Dietary Intake of Magnesium Page ) 25 28 3O 31 31; 37 38 39 In La La LIST OF FIGURES FIGURE Page 1. Graph Showing Variation in Serum Magnesium During the Day . . . . . . . . . . . . . . . . . 32 Graph showing Relationship of Blood Hagnesium and the Percent Deviation from Average Body Weight of AB Women . . . . . . . . . . . . . . . LS Graph showing Relationship of Magnesium Content of Urine and the Percent Deviation from Average Body Weight for AB'Women . . . . . . . . . . . . . . . h8 The Relationship of Urinary Magnesium and Serum Magnesium for Overweight, Underweight, and Aver- age Weight SUbjeCtS o o o o o o o o o o o o o o 0 SO IhTHODUCTIQh Experimental magnesium deficiency in rats was demon- strated first by Kruse et al in 1932; following this, biochemical studies have shown that magnesium plays an essential role in many enzyme systems. iapnesium is a constituent of enzymes of the Vitamin B complex and is an activator of several enzyme systems, as, for instance, the phosphatase enzyme (Stearns, 1951.) In a study of MAO patholoeical cases, Haury and Canta— row (lQhZ) found that the concentration of blood magnesium of arteriosclerotic and cardiac subjects tended to be higher than for healthy persons, although the range of values was similar for the two groups. These results suggested that magnesium might be involved in obesity since it has been reported that obese individuals show a greater tendency to develop cardiac disturbances than do people of averare weight (Downes, 1953.) That obesity is a sinnificant health problem in the United States is well known. Several metabolic studies have been undertaken in the attempt to understand this prob- lem (Brewer et a1, 1952; Young, 1952; Leverton and dram, 1951; Brown et a1, lth.) In 1951 Brown and Beerstecher stud- ied fourteen metabolic factors in the fastins urine of 10 overweight and in 10 underweight adult men. They demonstrated that the modified creatinine coefficient, pigment to creatinine ratio, urinary phosphate excretion and urinary calcium excretion were sufficiently different in the two groups to suggest that metabolic factors might be involved in obesity. Since mannesium is an important constituent of enzyme systems, the following study was planned to investi- gate possible relationships of the mannesium content of serum and urine of women to_body weight. The fasting blood serum magnesium and urinary excretion of mannesium were determined for 13 women of less than averare weight, for 13 women of more than average weight and for 22 women of average weight. thIbW 0P LITERATURE Distribution of Magnesium in the Body There is a paucity of information concerning the distribution of marnesium in the different tissues of the human body. According to Sherman (1952) magnesium com- prises 0.05 percent of the elements of which the human body is composed. This figure is based on a compilation of data from several sources. On this basis a man who weighs 75 kilograms would contain 38 grams of magnesium in his body. In an analysis of subjects who had died from acci- dents or diseases with no apparent disturbances in mineral metabolism Duckworth and Warnock (19h2-h3) reported that approximately 0.7 percent of the bone ash of the skeletons was composed of magnesium." This percentane differed widely for people of various ages. From the analysis of the magne- sium content of the skin (Brown, 1926) and of the heart (Cullen et a1, 1933; Wilkins and Cullen, 193h,) Duckworth and Warnock (19h2-h3) estimated that the value of mannesium in soft tissues was 8.2 grams per 70 kilocrams of body weirht. Duckworth and Warnock (19h2-h3) also compiled the magnesium contents of the soft and skeletal tissues for rirls from birth to 1h years of age and demonstrated that the skeletal mannesium tended to increase more rapidly than the marnesium content of soft tissues. At birth the ratio of skeletal magnesium to the magnesium in the soft tissue was two to one and at 1h years of are this ratio increased to a value of four to one. At the time of prepubertal growth, there was an increase in the estimated rate of deposition of magnesium in the tissues. The magnesium content of the body has been studied most extensively in the rat, but investigators have reported con- tradictory observations with respect to changes in magnesium composition with age. Buckner and Peter (1922) investigated magnesium content of crude ash of rats from two to he weeks of age, and found that there was little change in the magne- sium content with an increase in age. However, Medes and Humphrey (1927) studied the magnesium content of rats from birth to 150 days, and reported that the magnesium content of the body increased until the ninetieth day after which it remained constant. 0n the other hand, a rapid increase in the magnesium content of the bodies of rats from birth to four weeks was reported by Greenberg and Tufts (1936.) These workers found that the magnesium content of the rat carcasses remained constant from four to 11 weeks and after this age, there was approximately a 20 percent decrease in body magnesium. Possible differences in the body content of magnesium due to sex were suggested by Medes and Humphrey (1927) who showed that male rats contained a greater amount of magnesium in their bodies than female rats when compared on the basis of are. No significant difference was observed in the body content of magnesium of male and female rats when compared on a basis of weight. Buckner and Peter (1922) and Greenberg and Tufts (1936) reported that they had observed no marked differences in the content of mag- nesium in the bodies of male and female rats. Factors Affecting the Magnesium Content of Blood Within the last two decades, with the development of newer techniques for the analysis of magnesium in body fluids, many studies have been made of the distribution of magnesium in biological fluids. The following discussion is concerned with factors affecting the content of magnesium in blood Serum and plasma. A compilation of the blood magnesium values of apparent- ly healthy normal adults is presented in Table I. Values reported for only one or two individuals have not been in— cluded since the concentrations were within the ranee of those values cited. The values given in Table I range from 0.96 milligrams per 100 milliliters (0.8 milliequivalents per liter) to 3.51 milligrams per 100 milliliters; the average value is approximately 2.20 milligrams per 100 milliliters. Blood samples were withdrawn during unSpecified times of the day, except where noted. Whether or not the sample was venous or capillary blood was not indicated in most cases. o.H I >.H monE .Sdpom wcapmwh mmoa .mmazHA Q24 zomm>Afipmz Hm.m masovwmoh anon Gammonzm MH.N mpnoUSpm opfipwc cwa>wpwm qummHm :JOH .¢2mmfipwn I Essen omad .mmmHde mm.H ma.m I Ho.a mammflm eouficapmgon omoa .Hu no maHsm owwno>< owedm owwno>< omcwh H\uoa Hsooh\wfi OHQEdw no mhsuwz nonmwapuobaH cooam Ho psopaoo asfimocwwz mAHQzH Mmaq¢mm mom mmamommm mflqu> Smemzug 903m .mo zomHmdeoo < H mamdfi Few investigations have been reported on the blood magnesium values with regard to sex. Simonsen et a1 (19h7) found that the average serum mamnesium values for 21 female adults was 1.98 milligrams percent and that for 21 adult males, 2.06 milligrams percent. Bogert and Plass (1923) gave an average of 2.3 milligrams percent for the blood magnesium content for women; and Underhill and Dimick (1923-2u) reported an average of 2.7 milligrams percent for adult females. Salveson and Linder (1923) found a range of 1.7 to 1.9 milligrams percent for blood magnesium for seven adult males. The diurnal variations of serum magnesium have been studied by several investigators. Watchorn (1929) demon- strated that there were no significant differences in the values of blood mafinesium throughoht the day "...provided violent exercise is not taken..." However, it has been ob- served that cows show a significant diurnal rhythm with the lowest peak at 9 A.M. (Blosser et al, 1951.) No marked changes were noticed when blood magnesium of cows was analyzed at 9 A.M. for nine consecutive days (Blosser et a1, 1951.) Hald (1933) determined the fasting magnesium values of one person for three different days, and reported no marked changes in concentrations. Diverse results have been reported in several publications concerning the effect of temperature on he blood content of magnesium of different animals. Pleshtizer (1936) reported that in man, temperature had but a slight effect on the blood magnesium values. However, Pal et a1 (lghg) found that the blood magnesium values of cattle were higher in summer than in the winter. Suomalainen (1938) noted that the serum mannesium values in the hedgehog were three times larger during winter hibernation than in the summer waking state; this difference may have been due to temperature or to a variation in metabolic activity. Few studies have been made of the blood magnesium con- tent of peoples of different races. In 1950 Squires reported a wider range of blood masnesium values for adult men of Buchuanaland, South Africa than for white men of the same region. Nevertheless, he obtained an average of 1.71 milligrams percent for the South Africans, which agreed .satisfactorily with the averages reported for members of the white race. Radsma (19hh) recorded averaee values of 3.02 milligrams percent for native Batavian students, 2.81 milligrams percent for European born residents, and 2.13 milligrams percent for Batavian native servants. Studies of the variations in values for blood magnesium during the various stages of pregnancy have been inconclusive. Underhill and Dimick (1923—2h) reported similar values for blood magnesium for women in various stages of pregnancy. These workers found an averaae blood magnesium value of 2.7 milli- grams percent for normal non-precnant women. At the fourth a.“ run A Anh~ Jt.‘ C A. ‘w .fi 3. a ‘ and . v A. . h. I Ax~ n- as n y “71““ '~ WI 5 «a. r. N§U he I. . W. F: and sixth months of pregnancy the blood maonesium content was 2.0 milligrams percent. A slightly higher blood may- nesium value of 2.8 milligrams percent was observed during the seventh month of pregnancy and two days post-partum. That blood magnesium decreases slightly during menstruation has been reported by Nava (1950.) Bogert and Plass (1923) reported that there was little difference between the mag- nesium content of the umbilical blood of the newborn child and that of the venous blood of the mother at birth. Ac- cording to Miller (19Lh) values obtained for children tended to be within the upper ranpes of values reported for adults. Subnormal values of serum mavnesium have been re- corded both in cases of inanition and in cases which show a large increase in body weipht (Mellinghoff and van Lessen, 19h9.) In 19h? Sunderman reported that the serum marnesium concentration was above averare on the forty—fifth day of a voluntary fast by one man. 0n the fifty day after the cessation of the fast, the value was subnormal, but the serum magnesium returned to averare by the forty-third day after the fast. The tendency of serum magnesium to rise in the early part of fasting, followed by a drop with a rapid return to normal on refeeding also was observed by Morgulis (1928) in studies with dogs. No changes in the blood magnesium values were found during a seven day fast of the hedgehog (Suomalainen, 1939.) 10 The magnesium content of blood of pathological cases has been shown to be within the normal range. In l9h0-h1 Berstein and Simkins reported a slight increase in serum magnesium in cases with hypertensive heart diseases. In patients with arteriosclerosis with no hypertensive dis- orders a slight increase also was observed; in individuals with chronic nephritis and renal insufficiency, serum magnesium was elevated to two times the normal values.‘ In l9hh Miller recorded a case of tetany with a low plasma magnesium value. The subject was a child with osteo— chrondritis of the capital epiphysis of the femur. This tetany was cured by the administration of magnesium. Hirschfelder (193h) observed cases of convulsions and twitch— ing associated with low plasma magnesium values. Lower blood magnesium values have been reported for patients with bronchial asthma (Haury, 19h0.) In 19h2 Haury and Cantarow published a report on the serum magnesium values of th pathological patients. High serum magnesium values were recorded in individuals suffer- ing with chronic nephritis, arteriosclerosis with hyper- tension and hepatic disorders. Low values were observed -. for patients with toxic thyroid gland and polyfilandular wis- C" turbances, vasospastic di eases, malignant neoplasw, toxemia of pregnancy and epilepsy. Variable data were obtained from subjects with diabetes mellitis and orthopedic surgical Conditions. Serum magnesium values for individuals with ll endocrine disturbances showed no tendency to deviate from the normal range. From these results, the authors suggested that a low blood marnesium value for a patient might indi— cate a spasticity of the smooth muscles in tie blood vessels, or might be associated with a delay in callus formation, and tnat a hinher value for blood.ma~nesium minht be sud- gestive of the production of intra—articular adhesion and ossification. Factors Affecting Magnesium Metabolism In a review of mannesium metabolism, Cantarow and Trumper (lQMg) stated that about 50 to 80 percent of the total magnesium output of the body is excreted through the feces; he remaininh 30 percent is eliminated by the kidney. In an investiration of the urinary excretion of macnesium of 57 patients on admission to the hospital, Berstein and Simkins (19h0—hl) reported tlat the average daily excretion ranged from 17.3 milligrams magnesium to 285.0 millifirams magnesium per day, with an averare of 105.5 milligrams per day. These results are comparable to those reported by Walker and Walker (1935-36) who found that the average daily urinary excretion of magnesium was 103 milligrams with a range of 32.5 to 307.0 milligrams in a group of normal adults; the urinary eXcretion of magnesium for medical and surgical patients ranged from five to 2&3 milligrams per day with an average of 86 milligrams per day. In 1937-38 Weber recorded that the averane daily urinary excretion of magnesium ranted from 83.9 to 132.1 milligrams magnesium in eiqht young adults. The influence of various dietary factors on mannesium retention and urinary excretion of marnesium has been studied. Several investirations have demonstrated that mannesium, fed in the form of salts such as citrate (Bogart and McKittrick, 1922,) lactate (Carswell and Winter, 1931,) and sulfate (Hart and Steenbock, 1913) seems to be readily absorbed by the intestinal tract. According to Tibbets and Aub (1937) an increase in intake of ammonium chloride resulted in an increase in urinary excretion of magnesium. The studies indicate that an increased absorption might occur in an acid medium. Substances containing oxalates, benzoates and phy— tates have been reported to produce disturbiné effects on magnesium absorption (Stearns, 1951.) However, an investina- tion conducted by Walker et al in 1938 showed that man might adjust himself to a deficiency of magnesium induced by an increased dietary phytate intake. In a study of the reten— tion of magnesium of four normal men on a high phytate diet, a negative balance was observed within a few days after the subjects were on the diet. Magnesium equilibrium was attained after a period from two to 22 days. In the early part of the century, the addition of raw and dried milk to the normal diet of adult humans was found to increase the urinary excretion of magnesium (Givens, 1918.) In 19h2 McCance et al reported that a high protein diet 13 increased the absorption and therefore the urinary excretion of magnesium. The relative effects of starch, lactose, sucrose, and of vitamin D on mineral metabolism of rats was studied by Outhouse et al in 1938. These workers reported that lactose stimulated the excretion of urinary magnesium more than did the other saccharides. The amount of macnesium excreted in the urine showed a close correlation with the amount of magnesium ingested. Administration of cod liver oil did not significantly increase the retention of mannesium. Analyses of bone ash indicated that macnesium retention varied incon— sistently in relation to the maynesium content of the bones. The authors suggested that magnesium might be stored in the soft tissue. However Duckworth et a1 (l9h0) demonstrated in rats that the skeleton may act as a mobile reserve of mag— nesium in the time of dietary need. l The influence of minerals on magnesium absorption has been reported by De and Basu (10h9.) They found that an increase in the diet of calcium, phosphorous, iron, copper or manganese decreased the retention of magnesium to a measurable although slight extent. The favorable effect of the addition of three and one half cups of orange juice to the diet of two rirls, 10 and 11 years of age on magnesium retention was demonstrated by Chaney and Blunt (1925;) Coons and Coons (1935) reported a slight increase in magnesium retention in pregnancy when cod liver oil and wheat germ were added to the diet. It appears then, that an acid medium, lactose and certain of the vitamins seem to exert a favorable effect on magnesium absorption, whereas, starch, sucrose, the presence of certain minerals and the benzoates, phytates and oxalates produce an unfavorable action. Further studies are needed for a clearer understanding of the conditions which affect magnesium absorption. The possible interrelationship of calcium and mad- nesium in metabolism was reported first by Meltzer and Auer (1906.) These workers showed that the injection of calcium salts counteracted the effect of anesthesia which could be produced by the injection of magnesium salts. Mendel and Benedict (1909-10) found that the parental injections of either magnesium or calcium increased the urinary excretion of both substances. In 1937 Tibbetts and Aub stated that magnesium lactate injections caused an increase in the urinary and fecal excretion of calcium, and an increase hi! magnesium excretion. The influence of the inrestion of ammonium chloride and of high inorganic and orranic phos- phates on the retention of mafinesium and calcium also was investigated. Ammonium chloride resulted in a rapid elimina— tion of magnesium and calcium from the bones, but the phosphates (apparently had no effect on magnesium or calcium excretion. Tibbetts and Aub (1937) performed further studies on the effects of exopthalmic goiter and steatorrhea on maqnesium metabolism. Patients with these diseases are known to have large calcium excretions. However, no variations in manne- sium excretion and blood magnesium values were observed in these studies. The experimenters thereby concluded that magnesium and calcium, though closely related chemically, react differently in the body, and that magnesium is little influenced by the factors which affect calcium excretion. The effect of calcium and phOSphorous intakes on magnesium metabolism was studied on nine healthy college women (Leichsenrinp et al, 1951.) Results showed that urinary mannesium was significantly correlated with the intake of both calcium and phosphorous. However, there was no signi— ficant relationship between fecal calcium and phOSphorous with fecal mapnesium thourh the relationship between fecal calcium and phosphorous was significant. A lack of rela- tionship between fecal calcium and phosphorous with fecal magnesium also was reported. Isolated cases of tetany associated with low blood magnesium values also have suggested a possible interrela- tionship of calcium and mannesium. Kruse et al (1932) ob- served symptoms of hyperexcitability induced by magnesium .deprivation; this was a form of tetany which is associated with a normal blood calcium. Miller (19kt) also described a case of tetany in a patient who had a low blood magnesium. 16 A recommended daily allowance for mannesium has not been established by the Foods and Nutrition Board of the National Research Council. A suepested magnesium re- quirement for adults based on the data of Tibbetts and Aub (1937) was 200 to 300 milligrams per day for males with a ten percent reduction for females. Daniels and Everson (1936) estimated.that the marnesium requirement for children was 13 milligrams per kilogram body weight. Wang et a1 (1936) reported that the mannesium requirement of the adolescent female was 300 milligrams of matnesium per day. The data of Coons and Coons (1935) sugfested that 350 to h3O milliarams per day are required for pregnant women. Distribution of Kawnesium in Foods Relatively few investigations of the mannesium con- tent of foods have been published (Holmes et al, 19h8; Tilt and Hubbell, 1930; Clouse, l9h3; Kramer and Satterfield, 19h3; Toscani, l9h5; Peterson and Heppert, 1925, and Sherman, 1952.) .Studies which have been reported indicate that the best sources of magnesium are the protein rich foods such as Hulk, cereals, meats, and legumes. A compilation of mag— nesium values for individual food items has been made by Sherman (1952) and this table has been used widely in the evaluation of the magnesium content of dietaries (Kacy, 19h2.) 17 In a study of the chemical composition of 22 common foods, dummel et al,(l9h2) reported that laboratory analyses of the madnesium content of the particular foods tested agreed satisfactorily with values reported by Sherman. In a survey by the Carneeie Trust of the United King- dom (Duckworth and Uarnock, l9h2-h3) in which over 1000 urban and rural families were studied, vegetables and cereals seemed to be the main sources of madnesium in the diets of five socio-economic groups studied. However, it was shown that as the socio-economic status of the groups increased, a greater proportion of dietary sources of magnesium was obtained from meats, fish and fruits, and a lesser propor- tion of the dietary sources was obtained from cereals and vegetables. This survey showed that the lower income groups; representing one third of the total number of people parti- cipating in this investigation, had marnesium intakes that were deficient or on the borderline, accordinf to the esti- mated requirement of 250 milligrams per day for adult males and 220 milligrams per day for adult females (Tibbetts and Aub, 1937.) Sherman (1952) reported that in an examination of 150 American dietaries, the quantity of marnesium con- sumed per day ranged from lhO to 670 millicrams per day. Because of the wide distribution of this element, it is generally assumed that humans are able to incorporate a sufficient daily dietary supply, providing that the over-all food intake is ample (Stearns, 195 .) 18 Chemical Methods (Among the methods for the determination of maenesium in biological fluids, the phOSphate and the hydroxyquinoline methods have been the most commonly used. In 1909-10, McCrudden described a pravimetric procedure in which manne- sium was precipitated as 1.:qazit_ro}i.6a120, and the innited pyrophosphate residue was weiched. This method was adapted to the analysis of mannesium in foods, urine and feces. In 1922 both Brigms and Denis published colorimetric methods of measuring MgNHhPOh.6H2O by the production of a blue color with a molybdate salt. Berg (1927) introduced a method for the analysis of magnesium based on the principle that magnesium-eicht- hydroxyquinoline is precipitated in a hot ammoniacal solution. In 1932 Greenbera and Mackay developed a method of brominating the hydroxyquinoline and titrating the excess bromine with sodium thiosulfate. Yoshimatsu (1929) determined the hydroxyquinoline colorimetrically by using the Folin phenol reagent to produce a blue color. In 1937, Hoffman developed another colorimetric procedure for reading the hydroxyquinoline precipitate, which was based on the principle that the hydroxyquinoline combines with the ferric ions in a weak hydrochloric acid solution to form a hreen- blue color. 19 Both the phosphate and hydroxyquinoline methods for the determination of maenesium require the elimination of calcium as calcium oxalate; both methods are relatively time-consuming. In addition, the phosphate method may be criticized since the molybdate salt is relatively unstable, and the hydroxyquinoline method may be criticized since loss of precipitate may occur in carrying out the procedure. A simpler and, at present, more widely used method, for the determination of magnesium in biological fluids is based on the use of a dye, titan yellow, which becomes red in an alkaline medium, thus forming a stable magnesium- dye-lake. This observation was first made by Kolthoff in 1927, and it was adapted to the analysis of biological fluids by Hirschfelder and Serles in 193h. In this pro- cedure starch was used as a stabilizing agent for the mamnesium-dye-lake. In l9h6, Garner reported a methodology study on factors affecting the determination of blood mad- nesium. He found that the concentration of calcium, as it occurs normally in the blood, did not interfere in the determination of magnesium with the titan yellow method. He also observed that the unprecipitated serum proteins (Hirschfelder and Serles, l93h) created a large error. Garner introduced gum ghatti as a more suitable dispersing agent than starch. Hydroxylamine hydrochloride was found to be a successful stabilizing scent by Kunkel et a1 (19u7,) 20 who also reported that calcium and phOSphorous do not occur in blood in concentrations great enourh to interfere in the production of a stable red magnesium complex. Within the last decade, micro-methods for the deter- mination of different substances in body fluids by photo- electric or spectrophotometric procedures have been developed extensively. In 19h? Kirk et a1 devised a horizontal cuvette with a light path ten times that of the five cubic milli- meter cuvettes customarily used. Orange and Rhein (1951) adapted the use of this horizontal cuvette to the micro- determination of magnesium, since the concentration of mannesium was found to vary inversely with the length of the light path. Thereby, one-tenth milliliter of serum could be used in a single determination, rather than the two to four milliliters of serum which were required in previous methods. In this micro-method, polyvinyl alcohol was used as the diSpersing agent. More recently a spectrographic method has been described by Smith et a1 (1950) and a flame spectrOSCOpy procedure by Kapuscinki et al (1952.) However these methods are not well suited to the determination of mannesium in blood ‘because of the relatively low intensity of the lines in the magnesium spectrum. EXPERIMENTAL PROCEDURE Experimental Plan Subjects of this study were women who were considered to be of average body weight in relation to height and age, above average weight and less than average weight. Estimations of variations from.average body weight were made according to the Metropolitan Life Insurance Tables as reported by Cooper et a1 (19h?) for women from 18 to 29 years of age and as reported by Sherman (1952) for women 30 years and above. Ages of the subjects ranged from 18 to 53 years. The subjects were asked to come to the laboratory in the early morning, before breakfast. The subjects were to urinate immediately after rising; one hour later each subject was asked to collect a urine sample, emptying the bladder as completely as possible. This urine sample represented one-hour urinary excretion, collected by the subject in a fasting state. During this period, a capil- lary blood sample, approximately one milliliter in volume, 'was collected from a finger prick. The blood was centri- fuged and the serum removed for chemical analysis. The urine and serum were analyzed for magnesium. 22 A recall record of the diet eaten on the day previous to the collection of the urine and serum samples was ob- tained from each subject. The dietary intake of magnesium was calculated according to the values included in the com- pilation of magnesium content of foods reported by Sherman (1952.) Certain corollary investigations were carried out to provide data to help interpret variations in serum and urinary magnesium values among the subjects. The variations of the magnesium content of blood serum throughout the day were studied. Three women of average body weight in rela- tion to height and age were subjects for this series. Blood samples were obtained at 7:30 A.H. (fasting state), 10:00 A.M., 12:00 noon, 3:00 P.M., 6:00 P.M. and at 9:00 P.M. Magnesium content of the blood serum was analyzed. A study also was made of the possible variations in serum and urinary magnesium for individual women. Seven healthy women of average weight in relation to body height and age were subjects. Blood samples and one-hour urine excretions were collected at weekly intervals for a period of three weeks. The magnesium content of the serum and urine was determined. A comparison also was made of the magnesium.content of serum obtained from venous blood and that obtained from capillary blood. Subjects of this study were subjects of a 23 metabolic study* in progress in the Department of Foods and Nutrition at the same time that the present investi- gation was made. Nagnesium Content of Blood Serum The method of Orange and Rhein (1951) was used to esti— mate the concentration of magnesium in blood serum. Capil- lary blood was obtained from the finger by pin-prick and collected in small tubes, allowed to stand for an hour or more, then centrifuged at 3000 revolutions per minute for ten or more minutes. Duplicate samples of 0.1 milliliter of serum were pipetted into a three milliliter centrifuge tube. One and one-tenth milliliter of 10 percent trichloro- acetic acid was added rapidly. The solutions were mixed by holding the small tube against a stirring rod rotated by an electric motor, and then were centrifuged for ten minutes at 3000 revolutions per minute. The clear filtrate was transferred into a three milliliter centrifuge tube from which one milliliter of filtrate was pipetted and delivered into a five milliliter volumetric flask. This was followed by the successive additions of one milliliter of 0.1 percent polyvinyl alcohol, and one milliliter of 7.5 milligram percent of titan yellow solution. The solution was made to volume With 7.5 percent sodium hydroxide, mixed thoroughly and ‘ * Acknowledgment is made to Betty Hawthorne for permission to use these subjects for this study. transferred to horizontal cuvettes*, 50 millimeters in length. Optical density was measured by means of the Beckman Spectrophotometer at a wavelength of 550 milli- microns. Standard solutions of magnesium were prepared from magnesium ammonium phOSphate in concentrations Which ranged from one to five micrograms of magnesium per five milliliters of solution. The reading range was from 0.000 to 0.700 on the optical density scale. The procedure for the determination of serum mag- nesium was checked by the recovery of a known amount of 'magnesium from serum. Values for various amounts of added magnesium for three sera samples are given in Table II. Recoveries of magnesium averaged 99.86:0.66 percent. At the beginning of the study it was necessary to freeze some samples of serum for later analysis. ‘No sam- ples were held in the frozen state for more than a two months period. Control samples of serum were analyzed before and after frozen storage for two months and no change in magnesium content was observed. _‘* Microchemical Specialties 00., Berkeley, California. 25 TABLE II THE RECOVERY OF ADDED MAGNESIUM FROM BLOOD SERUM Sample Magnesium Magnesium Magnesium Magnesium Recovery Content Added Present* by of of Serum Analysis Magnesium mg/lOOml mg/ml mg/ml mg/ml % 1 .58 .50 1.08 1.08 100.00 " .50 1.08 1.08 100.00 " 1.50 2.08 2.08 100.00 " 1.50 2.08 2.08 100.00 " 2.50 3.08 3.16 102.50 2 .60 .50 1.10 1.10 100.00 " 1.50 2.10 2.09 98.66 " 2.50 3.10 3.05 98.00 3 .51 .50 1.01 1.01 100.00 " 1.50 2.01 1.97 95.1u " 1.50 2.01 2.00 97.00 " 2.50 3.01 3.17 103.00 Average. 0 O o o o o o o o 0 99.86 Stmdard D.v1&t10n O O O O O O O O 0 O O O O O O O O i 0.66 * By summation 26 Magnesium Content of Urine The macro—method for the determination of magnesium published by Garner (19h6) was adapted for the analyses of magnesium in urine. . If there was visible precipitate or blood in the urine, the material was filtered with ash-free Whatman filter paper, number forty. The urine filtrate was acidified with concentrated sulfuric acid and either analyzed immediately or frozen and held for later analysis. In the case of fro- zen urine samples, the urine was allowed to thaw in the refrigerator overnight. The following morning the samples were allowed to stand at room temperature for a few hours and were mixed thoroughly. The urine was diluted with distilled water. One milli- liter of 0.1 percent polyvinyl alcohol was added to eight milliliters of diluted urine, followed by 1.5 milliliters of 0.05percent of titan yellow solution, and two milliliters of four normal sodium hydroxide. ,Solutions were mixed thoroughly and the percent transmission was measured in the Coleman Spectrophotometer in square matched cuvettes. The reading range was from 70 to 100 percentage transmission. Working standards of magnesium were prepared from magnesium sulfate in concentrations which ranged from one to five grams of magnesium per 100 milliliters of solution. The method for the determination of magnesium was checked 27 by the recovery of known quantities of magnesium from urine; values obtained are reported in Table III. Re- coveries of added magnesium to urine averaged 99.77:,h.85 percent. The effect of freezing on the magnesium content of urine was studied by analyzing the magnesium of urine before freezing and after two weeks of frozen storage. No change in the magnesium content was observed. 28 TABLE III THE RECOVERY OF ADDED MAGNESIUM FROM URINE Sample Magnesium Magnesium Magnesium Magnesium Recovery Content Added Presentw by of Q of Urine Analysis Magnesium mg mg mg mg 74 1 .0026 .01 .0126 .0120 95.23 " .02 .0226 .0203 89.87 2 .OhZh .01 .0524 .0520 99.24 ” .02 .062u .0600 96.15 3 .0259 .01 .0359 .0355 98.89 " .02 .Ou59 .Oh50 98.0u h .0065 .01 .0165 .01u8 89.70 .0300 .01 .OAOO .OMZO 105.00 5 .0230 .01 .0330 .0310 93.9u .0335 .01 .0035 .ouua 101.61 Average. 0 o o 0 00000009607? StandardDeV1ationooo o o o o o 0.0 o o o 00 eta-085 a By summation 29 RESULTs Ann LIBC'SSION Variations in Serum and Urinary Magnesium in Individuals The magnesium content of serum from venous blood and from capillary blood samples from four subjects is given in Table IV. Values for serum magnesium from venous blood agreed closely with values for the magnesium content of serum from capillary blood. The average value for serum magnesium from venous blood for the four subjects was 1.16 milligrams per 100 milliliters and the average value for Serum magnesium from capillary blood was 1.17 milligrams per 100 milliliters. Dif— ferences between the magnesium content of venous blood serum and capillary blood serum for the individual subjects ranged from 0.00 to 0.04 milligrams per 100 milliliters. The daily variations in serum magnesium content for three subjects of average body weight in relation to height and age are presented in Table V. These values for serum magnesi' are plotted in Figure 1 in relation to the time of day duri; which the blood samples were obtained. There was not a con- sistent rhythm in serum magnesium.values for the three sub- jects. All three individuals showed a high serum magnesium concentration at 10 A.M. in comparison to the serum magnesium values obtained in the fasting state. The serum magnesium values for the three subjects were relatively constant at 12 noon, and at 9 P.M. The greatest daily change in serum 30 TABLE IV A COMPARISON OF VENOUS AND CAPILLARY SERUM MAGNESIUM Serum Magnesium Subject Venous Blood Capillary Blood mg/lOOml mg/lOOml CM 1.27 1.28 ES 0.98 ' 1.02 BHA 1.19 1.18 FK 1.18 1.18 31 TABLE Af- DIUBNAL RHYTHM OF SEMUM mAGnESIUM Serum Magnesium Subject Fasting 10AM 12PM 3PM 6PM 9PM ms ms/ ms/ ms/ ma/ lOle 100ml lOle 100ml 100ml NW 1.93 2.08 1.7M 1.75 1.83 1.73 NMA 1.36 1.52 1.30 1.36 0.94 1.35 BB .80 1.18 1.38 1.29 1.53 1.u0 32 >3 use uziao 22323: 221mm 2. 22.2.3) 3.326 1.25. .n 232... >45 “.0 mic. 32.55. b P P D 4 .1 4 4 saw in l _ Em . l 2.3 .22. cine“ '1 All! zz< Soo pnwaoz oceansm pcoowmm esaecapm unwaoz unmaom om< noofiasm meomhmbm Emonszo mo ZOHEHmome don—Hmwmm HH> mamgw 38 Hm.a m.o u o.oma o.saa 0.:0 mm Hz: a0.a m.o u o.o:a o.sma o.s0 em mm om.a 5.0 n o.0ma o.maH 0.:0 am 02 om.a s.0 u c.0ma o.maa 0.:0 Hm ez H:.H 0.0 n 0. HA 0. OH 0.00 0m an 9:.H o.0 u m. ma 0. Ha m.m0 Hm om s:.H o.H . m.maa :.:Ha m.H0 0H 22 m0.a o.o m.mma m.mma :.:0 an m: om.a :.m + o.sma o.oma m.m0 0m <22 sm.a :.m + o.mma c.0ma o.m0 mm 34 00.H 0.m + o.mma m.0ma 0.00 am m4 00.H 0.m + o.~:H 0.mma 0.00 om «m sm.H o.m + o.mma 0.5ma o.m0 am am H~.H 0.: + o.sma m.m:a o.s0 am am m0.a s.: + o.oma o.mma m.m0 m: on m0.H 0.: + 0.0ma o.mma 0.:0 mm HH om.a .s.m + o.mma o.oma o.m0 mm mm os.a m.0 + 0.:ma o.m:a o.m0 on me H0.H m.oa+ m.HmH o.mma m.mm mm 2e 00.H .HH+ c.0ma 0.oma 0.:0 mm .mo se.a H.mH+ o.mma s.mma m.m0 0m 32 as.a m.:a+ 0.0ma m.0:a m.m0 aa a: NE & and ana nofloca sumac; enmesapm «and Sean unwaom new m coaunsm cmwawwwo wmwwwuum nnwaoz unwaom om< noonnsm mBUMHmDm BmUHM3.mm4mm>4 mo ZOHBmHmome A mqmda 39 0m.a w.0mu o.mma m.:o 0.m0 mm so om.a .Hm- 0.0HH 0.ma o.om am 3n om.a 0.0m- 0.0HH 0.mo o.om am mm 0:.H :.oa- 0.0ma 0.moa H.00 0m 00 mm.a m.san 0.0HH o. o m.o0 mm ma Hm.a H.san 0.0ma :. Ha o.m0 mm as m:.a s.man o.oma p.0oa 0.:0 em o< mm.a m.mau o.0mH m.maa m.s0 ma ms 0m.H m.:a- 0.0HH H.Hoa o.o0 0m no NN.H m.:au 0.0:H o.sma 0.00 mm amm s .H 0.ma- c.0ma o.HHH m.m0 0: so om.a m.mau o.oma o.moa 0.00 :m 2m om.a o.oau o.sHH 0.:oa o.H0 :m as NE R and 09H monoca 000nm: enaecapm acne pnwaom pom a0n¢ coapaa>om unmaoz ooamwsm ”season enaecaum unwaoz pnwaom om¢ pecansm mBOMHmDm EmemzmeZD mo ZOHBmHmommm AdonMmm R mamOOG 03¢u>< 50¢“. tor—.(t‘o quucg m—C. 02¢ ied-23¢;- 00040 no 5.:u20_.r(..u¢ 08.32.; St‘KG .fl [IDS—l PIG-U; >00. da¢h>( iodt ZO—P(;IO rtuuflflb 8+ of. 3+ 3+ 3+ 3+ 3+ 2+ 0 2.. on. 2". C I m. md N n a u _ . . q. 1 % Q o A 3.. W . C. . C C v 00 o o 000 # o O o o. o F o o o. —o n 0 .MV a .2 u . _ m. co.“ made. blood magnesium values determined by other inves- tigators were presented in Table I and the ranve of blood magnesium concentration was 0.96 to 3.51 milligrams per 100 milliliters with an approximate averace of 2.20 milli- rrams per 100 milliliters. The fastinv serum macnesium values of the AB women in this study ranped from 0.92 to 1.00 millirrans per ltO milliliters with an averaae of 1.25 milligrams per 100 milliliters. The fasting serum magnesium values of the MB women were similar to the lower values for blood magnesium reported by other investigators. However the range of values reported in the literature was greater than the ranre observed with these subjects. In the investigation of the weekly variations of fastina serum magnesium, no sirnificant changes were observed in the serum magnesium values for three successive weeks. This indicated that the fasting serum marnesium values of women were relatively constant for individuals in a fasting condition over a period of several weeks. In contrast, the daily chances in serum maonesium for three individuals showed individual variations of 0.35: 0-58 and 0.73 milliqrams per 100 milliliters. These results suggest that the daily changes in serum magnesium values of individuals are subject to greater variations than the serum magnesium concentrations in individuals who are in a fasting condition. Since many of the blood marnesium in values reported in Table I were determined at intervals throughout the day, it might be expected that the pub- lished values would vary more widely than the values for serum mannesium which are reported here. Urinary magnesium. Tables X through XII present the magnesium values of one hour urine collections for the QB women. The average urinary magnesium excretion was 2.hh milligrams per hour. Table X gives the urinary magnesium values for the overweight subjects. The average value of urinary magnesium was 2.h5 : 1.13 milligrams per hour with a range of 0.62 to 3.92 milligrams per hour. The urinary magnesium values for the subjects Ofelverage body weight are presented in Table XI. The averape concentration of urinary mannesium was 2.32 :_l.2 with a ranpe of 0.70 to 5.17 milligrams per hour. Table XII records the urinary magnesium values for the underweight subjects. The averane value of \ urinary maenesium was 2.61 1 1.28 milligrams per hour with a range of 0.h7 to h.79 milligrams per hour. The relationship between urinary magnesium aid the percent deviation of body weipht from the standard weight was investigated. It is evident from the diagram in Figure 3 in which urinary magnesium is plotted against the percent deviation of body weight from standard weight, that there is a lack of relationship between these two factors. The is auto; 3 as. has! >93 3532 33.“. 3:553 .533“. m3... 02¢ 92:: no .53.»on 223.333 no 2322.542. 2:323 E<¢o ...&u¢:u.m . .2653 room uu<¢u>< 52.“. 22.553 hzmuxwm .2. on. So 3+ 3... 8+ 8. 20 o o... 8- 8.. _ . _ . . a o o a 0 v 0 O O n o n _, o u a O O 0 JW .v 0 . O b O Wt... ' WINS? NSVW XHVNifl fl correlation coefficient obtained for urinary magnesium of the subjects and the percent deviation of body weight from average was +0.069. This indicated that there was not a significant relationship between urinary magnesium and body weight. The relationship of urinary magnesium and the percent deviation of body weight from standard weight also was investigated in the three groups of women. There was not a significant relationship between these two factors in the overweight individuals, subjects of average body weight and underweight subjects. Possible differencdgin urinary magnesium of the over- weight subjects, subjects of average body weight and underweight subjects were investigated. According to the Fisher "t" test there was no significant difference in the urinary magnesium excretions of these three groups. These results are in agreement with the results obtained by Browr and Beerstecher (1951) who reported that they found no significant relationship in the fasting urinary excretioh of magnesium in 10 overweight and in 10 underweight adult men. A correlation coefficient of only +0.190 was obtained in the comparison of serum magnesium and urinary magnesium for the total group. This indicated that the relationship between urinary magnesium and serum magnesium was not signi— ficant. The relationship of urinary magnesium and serum magnesium for the three groups of overweight, average weight and underweight subjects also was investigated. Figure h 50 95263 .2633 35.23 23 ”522.532: .9225qu «on 323222. :5ch 92 523.532 5.523: no Stator—.59... m2... .c meant. s..\«... «.2322: >552: 0M 9t? 0% o.~ 9.. 030 o - r03 I I \I I O IIII|I.IIII|I"|I“|II.I O I i ’ J o u Inu‘ ill a o a o o o l I ‘ . O C U C o . n 5223392... row. . .2653 min: .- 5333-8 I u Pro-3:. pom/Bu wmnnvw mun 51 shows the regression of serum magnesium upon urinary marnesium ! D . , V f for the three aroups. A correlation coefficxent of +O.CoO ‘ (P=0.0S) was ootsined for the unxerwe‘ nt suojects for serum cated t-at V ere Ho masnesium and urin rv mannesium. This ind was a sifinificant relationship between serum mar:iesium and urinary magnesium in the underwei ht subjects. A correlation coefficient of +0. 083 and +0. 050 was obtained for tne over- weight subjects and the subjects of average body weight respectively oetvee n these two factors. Thus there was not a sirnificant relationship between serum magnesium and urinary magnesium in the overweisht subjects and in tie Sije cts of average body Joight The latter results tend to support the studies of Berstein and Simkins (lQLO-hl) who found no rela~ \ tionshir between blood esiu . and urinirf asynesiuu is / normal healthy individuals. It is of interest that a relationship of serum mannesium and urinary maynesium was observed among the underweight subjects but not among tie sub1ec cts of averafie or above average weight. This may be a chance observation; however, it also presents the possibility that the metabolic relation— sLip of serum and urinary magnesium may differ for the under weight individual from that of the individual of averare weight or the obese person. 52 Relationship of dietary magnesium to serum and urinary magnesium. The calculated magnesium content of the diets ) of overweight women is reported in Table X. The average } intake of magnesium was 212 milligrams per day with a range from 57 to 3h2 milligrams per day. The daily intakes of magnesium of subjects of average body weight are presented in Table XI. The range was from 1h? to h9h milligrams magnesium per day with an average intake of 264 milligrams of magnesium per day. Table XII records the magnesium con- tent of the dietaries of the underweight individuals. The average intake of magnesium was 273 milligrams per day with a range from 157 to h3h milligrams per day. The average magnesium intake for the total group was 253 milligrams per day. Table VI presents the daily magnesium intake of seven individuals of average body weight for three successive weeks. For the first day, there was an average intake of 229 milligrams magnesium. In the estimation of dietary magnesium for one day in the sedond week, an average intake of 263 milligrams per day was obtained, and for one day of the third week, an average intake of 250 milligrams magnesium per day was calculated. Individual variations in dietary intake of magnesium for the three weeks ranged from 36 to 1&9 milligrams magnesium per day. There was not a significant relationship between dietary magnesium and serum magnesium for the total group or for 53 the three groups of overweight subjects, subjects of average body weight and for the underweight subjects. This indicates that serum magnesium concentrations do not vary with the dietary intakes of magnesium. Daniels and Everson (1936) in a retention study had suggested that urinary magnesium might show a relationship to magnesium intake. However the urine samples of this study were collected over a period of one hour after the subject had been in a fasting condition for at least eight hours. Therefore magnesium intake in this case does not represent the utilization of the daily magnesium intake by the body. There was not a significant relationship between fasting urinary magnesium excretion and dietary magnesium intake (correlation coefficient, +0.0l6.) The studies of serum and urinary magnesium which have been presented here have indicated that the magnesium con- centration in blood serum is relatively constant. Although variations in serum magnesium from 0.35 to 0.73 milligrams per 100 milliliters of serum were observed for three sub- jects at different times of the day, these variations are relatively small; moreover, very little variation was ob- served in.fasting serum magnesium for seven subjects for three successive weeks. The range of serum magnesium con- centration for the subjects of different body weights was small and no correlation was found between fasting serum magnesium and the estimated dietary intake of magnesium. Sh These observations suggest that serum magnesium may be maintained at a relatively constant concentration by certain metabolic factors. Relatively little is known concerning metabolic factors which influence serum magnesium. Cantarow and Trumper (lQMQ) reviewed factors affecting the regulation of serum magnesium and reported that serum magnesium was not greatly affected by the administration of phosphate, protein and vitamin D. The administration of the para- thyroid hormone caused a slight increase of:serum magne- sium. In oxalic acid poisoning, a decrease in serum calcium was associated with an increase in serum magnesium, and in the injection of magnesium salts, an increase in serum mag: nesium was accompanied by a decrease in serum calcium. It is apparent that there is need of further investigations on the metabolic factors which are involved in magnesium metabolism. SUl‘dlniil'iY A311) COL. ULUQ I bl": S The madnesium content of blood serum and urine of LB women was studied in relation to body weight. The afe range was from 18 to 53 years. All of the subjects were apparently healthy at the time in which they participated in this study. Fastinc blood serum was determined by the titan yellow method of Orance and Rhein (1951.) Urinary magnesium of a one hour urinary excretion, collected by subjects in the fasting state, was analyzed by the method of Garner (lth.) The magnesium.content of the diet for the day previous to the collection of blood and urine samples was estimated. The percent deviation of body weight from the averare weight was calculated from standard weight, height and are tables. Of the hB individuals studied, 13 were 20 percent or more overweinht; 13 were 10 percent or more underweight and 22 were within a ~10 to +15 percent of their average body weights. The average serum magnesium value for the group was 1.21 milligrams per 100 milliliters. The overweight subjects had an average serum marnesium value of 1.25 1 0.19 milligrams per 100 milliliters, and the underweight subjects 1.20:0.11 milligrams per 100 milliliters. Statistical analysis by the Fisher "t" test indicated that there were no differ- ences in serum magnesium for the three groups. The averafe urinary magnesium excretion for the total group was 2.uu milligrams per hour. The overweight subjects excreted an average of 2.45:1.13 milligrams magnesium per hour; the underweight subjects, 2.61:1.28 milligrams magne- sium per hour and the subjects of average body weight, 2.32:1.23 milligrams magnesium per hour. There was not a significant difference in urinary magnesium for the three groups. No significant relationship was found between serum magnesium and the percent deviation of body weight from the standard weight or between urinary magnesium and the percent deviation of body weight from standard weight for the subjects. A correlation coefficient of 0.660 (PrO.CS) indicated that there was a significant relationship between serum and urinary magnesium for the urderweight subjects. The relationship of these factors in the individuals of average body weight or for the underweight subjects was not significant. There was not a significant relationship be- tween serum magnesium or urinary magnesium and dietary mag- nesium for all subjects. A comparison of the magnesium content of serum of venous and capillary bloods showed no marked differences. A study of the weekly variations of fasting serum magnesium 57 and urinary magnesium for seven subjects demonstrated that there were no significant changes in serum magnesium values or urinary magnesium concentrations for three successive weeks. An investigation of the daily variations of serum magnesium showed that the individual variations of serum magnesium was 0.36, 0.58 and 0.73 milligrams per 100 milliliters. REFERENCES CITED Berg, R. 1927: Neue Wege zur Bestimmung und Trennung der Metalle mit Hilfe von o-Oxychinolin (II. Mitteilung.) Bestimmung und Trennung des Magnesiums Z. Anal. 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