TRANSFER OF ASCORBIG ACTD 3N A HEMODIALYZER POSSIBLE PROTETN BOUND ASCORBIC ACID ~~~~~ _ mommn'srm{imam} .: f. : *" JEAN maczxowsmauacz ' -- I .. 1971 ....... Ea; rwasw LIBRARY T Michigan State . University I ABSTRACT TRANSFER OF ASCORBIC ACID IN A HEMODIALYZER POSSIBLE PROTEIN BOUND ASCORBIC ACID By Jean Mieczkowski Burge A patient undergoing long-term renal dialysis may lose a number of nutrients from his blood. This is due to the fact that the dialysate solution contains only a limited number of nutrients. The constituents of the blood not present in this dialyzing solution may be removed from the blood during dialysis. According to other investigators folic acid is lost during hemodialysis whereas other B vitamins appear to be unaffected. Vitamin C has also been reported lost from the blood during hemodialysis (Sullivan 33 §;., 1970). The purpose of the present study was to evaluate the rates of transfer of ascorbic acid from blood to dialysate fluid across a dialysis membrane, and to determine whether some ascorbic acid is present in the blood as a non- diffusible complex. For the initial work a miniature Isr- allel flow hemodialyzer, Babb-Grimsrud modification of the Kiil dialyzer using foam nickel metal membrane support rather than the original plastic support, was used. By this means it was possible to recycle small blood samples by Jean Mieczkowski Burge means of a pump. The results of this phase of the study showed that the permeability of ascorbic acid in this sys- tem was 0.0106 cm/min, fifty five percent of the ascorbic acid was lost after three complete recyclings of human plasma. Additional recyclings resulted in no greater loss of ascorbic acid which did not dialyze despite repeated recyclings. This may represent a protein bound form of the vitamin. When anesthetized dogs were attached to a hemodialyzer the ascorbic acid in their blood drOpped during the first 30 minutes of dialysis and remained stable thereafter. There was no indication that the ascorbic acid level of the blood was being restored when the dialysis was continued for four hours. Results similar to the preceeding were obtained when blood samples were secured from patients undergoing hemo- dialysis at a local hOSpital. A rapid loss of ascorbic acid occurred during the first 25 minutes followed by a plateau, indicating a possible bound form of ascorbic acid in human plasma. Patients dialyzed 36 to 40 hours after the initial dialysis were not able to recuperate the levels of plasma ascorbic acid of the previous dialysis treatment when de- pending upon diet alone. TRANSFER OF ASCORBIC ACID IN A HEMODIALYZER POSSIBLE PROTEIN BOUND ASCORBIC ACID By Jean Mieczkowski Burge A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1971 ACKNOWLEDGEMENTS The author would like to express her sincere thanks to: Dr. Olaf Mickelsen for his guidance and help throughout this study Dr. Donald K. Anderson for the use of the hemodialyzer and for his technical assistance and advice Dr. Rachel Schemmel for her encouragement and assistance Drs. Dena Cederquist, W. D. Collings and T. Johnson for their aid and advice Dr. Martin Jones for his aid in obtaining data on patients under- going hemodialysis her husband, Charles for his encouragement and love 11 TABLE OF CONTENTS Page INTRODUCTION 0 . . . o o o o o . o o o . o o o . o . . l REVI EW OF L I TMTURE . C O O C C C O O O 0 O C O . O 3 DevelOpment 0f HemOdialySiS o o o o o o o o o o o o 3 Dialysis Membranes o o o o o o o o o o o o o o o o o 6 Dialysis Fluid . . . . . . . . . . . . . . . . . . 7 Applications for the Artificial Kidney . . . . . . . 8 Effect of Hemodialysis on the Nutritional Status Of the Individual 9 o o o o o o o o o o o o o o o o 10 Clearance of Vitamins by Dialysis . . . . . . . . . ll ASCOTbiC ACid o o o o o o o o o o o o o o o o o o o 12 Binding 0f Ascorbic ACid o o o o o o o o o o o o o o 14 OBJECTIVES OF PRESENT STUDY 0 . o o . . o o o o o o o 16 O O O O O S IJIETHODS O O O O O O O O O O O O O O O O O O Appalr‘atus . C . O . O O . . O . O . . O O O O O . . 17 In Vitro StUdieS o o o o o o o o o o o o o o o o o 17 IEZVlVO StUdieS o o o o o o o o o o o o o o o o o 18 AscorEic Acid and Potassium Chloride Solutions . . . 18 Plasma Samples . . . . . . . . . . . . . . . . . . . 21 subjeCts - Canine o o o o o o o o o o o o o o o o o 21 SUbjeCtS - Human o o o o o o o o o o o o o o o o o o 22 RESULTS AND DISCUSSION 0 O 0 O O O O O O O O O O O O 0 21+ Permeability of CuprOphane PT 150 Membrane to Ascorbic Acid . . . . . . . . . . . . . . . . . . . 24 Diffusion of Ascorbic Acid from Plasma - in vitro . 27 In Vivo StUdieS - Canine o o o o o o o o o o o o o o 33 I§:v1VO StUdieS ~ Human o o o o o o o o o o o o o o 33 SUMMARY 0 o o o o o o o o o o o o o o o o o o o o o o 42 BIBLIOGRAPHY o o o o o o o o o o o o o o o o o o o o o 43 APPENDIX 0 o o o o o o o o o o o o o o o o o o o o o o 48 iii Table 10 LIST OF TABLES Composition of Dialysate Fluid: Hemotrate Formu1a300000000000000coo Permeability of CuprOphane PT 150 Membrane to Ascorbic Acid and Potassium Chloride atZSoC.................. Permeability of Ascorbic Acid Through a CuprOphane PT 150 Membrane . . . . . . . . Ascorbic Acid Concentration (mg%) in Canine Plasma with each Pass Through the HemOdialyzer00000000000000. Ascorbic Acid Concentration (mg%) in Human PlaSma with each Pass Through the HemOdialyZ-ercoo-0.000000... Comparison of Two Methods of Ascorbic Acid Analysis in Human and Canine Plasma . . . . Ascorbic Acid Concentrations of Canine Plasma: In Vivo Passages Through a Parallel FlOWHemOEaIyzerooooooooooooo Medical History and Dietary Intakes of Patients Undergoing Hemodialysis . . . . . Plasma Ascorbic Acid Concentration of Hemodialysis Patients . . . . . . . . . . . The Determination of the Permeability of Ascorbic Acid Through a Hemodialyzer . . . iv Page 25 26 28 29 52 3A 36 38 49 Figure LIST OF FIGURES Chemical Configuration of L-ascorbic ACidoooooooooooooooooooo EXploded View of the Parallel Flow Hemodialyzer................ Open L00p System for Diffusion of Ascorbic ACidfromPlasma-oooooooooooooo Closed L00p System for Ascorbic Acid Permeability................ Ascorbic Acid Concentrations of Canine Plasma In Vitro Passages Through a Parallel Flow HemOdlaIyZer o o o o o o o o o o o o o Ascorbic Acid Concentrations of Human Plasma In Vitro Passages Through a Parallel Flow EamoaTaIyzer................ Ascorbic Acid Concentrations of Canine Plasma: In Vivo Passages Through a Hemodialyzer................ Plasma Ascorbic Acid Concentrations of Hemodialysis Patients During Dialysis: I . Plasma Ascorbic Acid Concentrations of Hemodialysis Patients During Dialysis: II . Page 12 20 21 21 30 30 31+ 39 40 INTRODUCTION Pitts (1968) defines uremia as "a complex of symptoms and signs reflecting the dysfunction of all organ systems based on failure of renal regulation of the composition and volume of the body fluids." The term "Uremia", urine in the blood, was first used by Piorry in 1840, te describe the consequences of the abnormal retention of excretory products. The uremic syndrome manifests itself in altera- tions in every major organ system of the body. Apathy, anorexia, nausea, pulmonary edema, anemia and osteomalacia are frequent symptoms of the uremic patient. According to Burton (1967) 50,000 peOple die each year from uremia. Nineteen percent of this pOpulation are be- tween the ages of 15-54 years, 79% are over 55. and 2% are under 1A years of age. With the technology and economic resources available today 6,000-10,000 of these patients 4 would be considered ideally suited for hemodialysis or kidney transplants; the two current modes of treatment for uremia (Leonard and Dedrick, 1968). Jones (1971) estimated that hemodialysis could prolong the lives of as many as 2#,000 of these patients. In a situation where financial limits to treatment have been removed, the demand for hemodialysis will exceed 40,000 persons by the year 1976 (Leonard and Dedrick, 1968). This estimate would not be altered by even the most extensive transplant program. 2 The prognosis for the hemodialysis patient in the first year is good, with an 85% survival rate, however, by ten years this has declined to 30%. Organ transplants, with dialysis as a backup treatment if the tranSplant fails, boosts this ten year prognosis to 75% (Burton, 1968). Knowledge regarding the effect of long term hemodialysis on the patient's nutrient stores becomes important with the ever increasing use of this type of maintenance; however, the research data available on the nutritional effect of hemodialysis are meager. Anemias due to iron and folic acid deficiencies have been reported in long term hemodialyzed patients (Comty gt al., 1968). Jones (1971) has observed petechiae and hematomas suggestive of an ascorbic acid deficiency in some of his patients undergoing hemodialysis. On the basis of the evidence associated with the re- lationship of ascorbic acid to iron and folic acid metabolism and since ascorbic acid is so soluble in water and has a relatively low molecular weight it seems justifiable to quantify the losses of ascorbic acid as a result of hemo- dialysis. The fact that ascorbic acid appears in the sweat only to a slight extent (Mitchell and Edman, 1951) may in- dicate that this vitamin exists at least to a certain extent in a non-diffusible form in body fluids. REVIEW OF LITERATURE Development 9§_Hemodialysis The hemodialyzer is a system in which circulating fluids are dialyzed across a semipermeable membrane against a coun- tercurrent circulating dialysate fluid, the composition of which can be changed to favor the removal of Specific sub- stances. The deve10pment of the first experimental dialyzer in 1913 by Abel, Rowntree and Turner created the theoretical possibility of prolonging life in patients with acute renal insufficiency. Kolff (1965) changed this to a practical possibility with the develOpment of the first successful artificial kidney (hemodialyzer) in 1944, for the relief of acute symptoms of uremia in human patients. Rehabilitation of patients with chronic renal insufficiency was hampered, however, by the limited number of blood vessel cannulation sites. Quinton 32 a1. (1960) overcame this problem with the develOpment of the Teflon-Silastic shunt and expanded the use of the artificial kidney to chronic uremic patients. Teflon silastic tubes are inserted into a major artery and a superficial vein of the patientis arm or leg, a teflon silastic shunt is connected to these tubes for uninter- rupted blood flow. Blood tubing connecting the patientis blood to the machine replace this shunt when the artificial 3 an jJi Olism Patie minint assem] m99t a H: the tw “’11 h. Zhe die held it The die L, kidney is in use. Although some problems do occur, many patients are able to maintain functional shunts for as long as two years, when another site of cannulation can be used. Brescia 22.2l' (1966) develOped a surgically created inter— nal fistula between the radial artery and a superficial vein as a method for repeated access to the circulation in patients undergoing maintenance hemodialysis. This method 7‘ solves many of the problems with infections, clotting and ‘ physical trauma inherent with the exterior arterio-venous shunt, and has remained functional for longer than two years. ‘ The use of this method is contra—indicated, however, for the patient over 40 years because of the increased incidence of stenosis in this age group. Freeman §t_gl.(l965) outlines seven characteristics of an ideal hemodialyzer: Removal of toxic products of metab- olism in an efficient manner; removal of water from oliguric patients by ultrafiltration; a low internal volume requiring minimal blood priming and reducing blood loss; simplicity of assembly; safety; and finally low initial and maintenance costs. None of the hemodialyzers currently in use clinically meet all of these requirements. The twin coil and the parallel flow hemodialyzer are the two major types of artificial kidney now used. The twin coil hemodialyzer was originally develOped by Kolff in 1944. The dialyzing unit consists of two parallel ce110phane tubes held in a fiberglass mesh and wrapped around a central core. The dialyzing surface and priming volume varies depending 5 upon the length of the ce110phane tubing from 0.9m2 to 1.9m2 and with a volume capacity from 520 to 1200 ml respectively. Satisfactory dialysis can be achieved in as little as six hours with this dialyzer if it contains the longer length of tubing. The coil is prepackaged and presterilized re- ducing the assembly time. However, priming volume and high resistance in this system, when the optimum dialysis time is met, necessitates transfusions and the use of a pump in- creasing the dangers inherent with transfusions and the possibility of hemolysis from the pump. Kiil in 1960 designed the parallel flow hemodialyzer; a low resistance system which eliminates the need for a pump and reduces the priming volume of blood to that which can be supplied by the patient's own blood system. The dialyzing unit consists of a blood layer between cellophane sheets sup- ported by polyprOpylene boards. The dialysate solutions cir- culate outside the ce110phane through grooves milled in the plastic boards. Its direction of flow is Opposite to that of the blood (Cole gt_§l., 1963). A two layer Kiil 2 and can be assembled dialyzer has a surface area of 1.12m in 45 minutes. Kuruvila gt_§l. (1969) compared the Kiil and the twin coil hemodialyzers in a clinical study of 58 patients and observed fewer incidences of hypertension and pulmonary edema, lower transfusion requirements and lower mortality associated with the Kiil hemodialyzer. The twin coil has, however, a greater efficiency in the removal of toxic solutes. This factor, along with its shorter assembly time, makes this machine preferred for emergency treatments or where rapid removal of toxic substances is essential (Freeman 33 31., 1965). Dialysis Membranes "A clinically acceptable membrane must be, all at once, a regulator of concentration - and - pressure - induced transport, a mechanical barrier and a surface which is compatible with the biological environment." (Wezmar and Leonard, 1970) The essential function of a hemodialysis membrane is to control the degree of intercommunication between the permeable components in the blood and dialyzing solution. A membrane is also a mechanical barrier, fixing the geometry through which blood and dialysate fluid flow. Cellulosic membranes, commercial films currently in clinical use for extracorporeal dialysis, primarily Operate by the diffusion of solute through microholes or pores (Klinkman gt_§l,, 1970). These membranes discriminate on the basis of size and shape, small ions and molecules permeating relatively easily while increasing size decreases the amount of transport. Alexander §t_§1. (1965) has shown that molecules with molecular weights as high as 16,000 to 19,000 can be transferred in a dialyzer. Cellulose membranes are neutral and have negligible ion exchange capac— ity at the ionic concentrations employed in hemodialysis (Leonard and Dedrick, 1968). 7 CuprOphane PT 150* and Visking# membranes of regene- rated cellulose are the two most widely used of the Cellulose membranes. CuprOphane membranes have an average wall thick- ness of approximately l6/Land show extreme evenness of pore size and distribution. The average pore size is from 10-15 R (Klinkman 23': 3;” 1965). Several investigators have at- tempted to modify or develOp new hemodialysis membranes to replace the commercial membranes. Although each has Specific characteristics which may be superior to the cellulose mem- branes, as yet none of the newer membranes has proven to be superior in overall characteristics. Dialysis Fluid The dialysate fluid is the one component of hemodialysis which can readily be changed to favor the removal or addi- tion of Specific substances. The composition of the dialy- sate solution must include certain essential chemicals in sufficient concentrations to maintain normal levels of these solutes in the blood of the patient undergoing hemodialysis. Sodium, acetate, calcium, magnesium, chloride, potassium and glucose are the essential chemicals (Freeman 23 21., 1965). Table 1 gives a typical composition of a dialysate fluid. Potassium, one of the essential chemicals, may be retained by the patient with chronic renal failure and consequently, -I- Bremberg Corporation #Dupont Corporation 8 Table 1. Composition of dialysate fluid: Hemotrate Formula 5a Chemical g/liter Ion meq/liter NaCl 5.5 Na+ 150.0 Na Acetate 4.7 Acetate 35.0 CaClZ‘ 2H20 0.18 Ca++ 2.5 MgClZ‘ 6H20 0.15 Mg++ 1.5 KCl 0.15 K+ 2.0 Dextrose 2.0 Cl” 101.0 aMceaw Laboratories hemodialysis concentrate (Cat. No. R-1625) after dilution 54:1. this solute may at times be deleted from the dialysate solu- tion in an attempt to lower the patient's elevated blood potassium. Other constituents of the dialysate solution may also be manipulated when clinical situations require it. Applications £9; the Artificial_Kidney Treatment of acute and chronic uremia far surpasses all other uses for the artificial kidney. Merril §t_§l. (1964) first demonstrated the feasibility of performing hemodialysis in the home. Failsafe systems have been develOped which monitor the patient's blood pressure and the machine for blood-to-bath leaks. This system will awaken a patient Should a problem arise, making unattended overnight dialysis possj hemoc patic six 1 thos comp. the I hemo cost medi leas possible. The training of the patient to perform home hemodialysis requires from four weeks to a year with many patients able to perform satisfactorily with an average of six weeks instruction (Pendras gt al., 1970). Although at present patient insurance will only cover those expenses incurred in the hospital, many insurance companies are now considering extending this coverage to the home dialysis patient. Presently the cost for home hemodialysis is 810,000 initially, followed by an annual cost of approximately 35,000 for supplies, maintenance and medical assistance. This figure would be increased by at least 5-5 fold had the patient remained hospitalized. Acute poisoning is the second most frequent use of hemodialysis. Hemodialysis is effective in quickly removing many toxic substances after they have been absorbed into the blood stream. Barbituate, salicylate and glutethemide are 'the three most common compounds requiring dialysis and have been shown to be effectively cleared from the blood thereby (Kiley. 1969). welder and Faillace began treatment of acute alcoholism with the hemodialyzer in 1969 (Med. Wld. News, 1969). Pre- liminary observations revealed complete recovery in six hours and a tendency to remain sober for an undefined length of time. These investigators also showed that alcohol in- duced symptoms of CNS stimulation; nausea, headaches and delerium tremens were completely eliminated. on t] ever (1964 meabl acid: dial; essel GXCGG value Posit conce weekl inves and 4c Patiex treat; tein c treatm time 0; stricte patient sodium J is the I 10 Effect 9; Hemodialysis gn_the Nutritional Status of 222 Individual Knowledge regarding the effect of long term hemodialysis on the patient's nutrient stores becomes important with the ever increasing use of this type of maintenance. Ginn 23 al,, (1968) has shown that all the essential amino acids are per- meable to the dialysis membrane and from 2.0 to 3.5g of amino acids were recovered from the dialysate after six hours of dialysis. Gulyassey §£_§1., (1968) also observed several essential amino acids in the dialysate, at concentrations exceeding 50% of the minimal daily requirements in normal man. 0.75g/kg body weight (50g) per day of high biological value protein is necessary to maintain a neutral or slightly positive nitrogen balance and to maintain serum albumin concentrations in patients undergoing hemodialysis twice weekly. Sorensen and KOpple (1968) as well as many other investigators have reported poor dietary adherance to the 20 and 40g protein diets. Pendras (1968) observed that his patients would willingly undergo an additional dialysis treatment (5/week from 2/week) in exchange for an 80g pro- tein diet. The therapeutic effect of a sodium restriction in the treatment of renal disease has been established since the time of the Kempner rice diet (1944). Sodium may be re- stricted to as little as 500mg per day in a severely uremic patient, however, the average hemodialysis patient has a sodium restriction of from 1000 to 2000 mg sodium, (5,000mg is the normal adult intake, Frank and Mickelsen, 1969). Sore I underg late 1 output Cleare well C 11 More recently potassium has been restricted in the patient undergoing hemodialysis. Dangerously high levels can accumu- late in these patients, especially those with no urinary output, between dialysis (Louis and Dolan, 1970). Clearance 2; Vitamins by Dialysis Anemias due to iron and folic acid deficiencies have been well documented in long term hemodialysis patients (Comty 23_ 31., 1968; Hampers §t_a;., 1967). Hegstrum.g£‘§1. (1961) reported develOpment of perepheral neurOpathy and symptoms of a pantothenic acid deficiency in chronic uremic patients undergoing hemodialysis. No plasma vitamin levels were measured. Lasker 23.21. (1963) studied the levels of the B vitamins in patients undergoing peritoneal dialysis and re- ported a decline in folic acid after dialysis. Low nicotinic acid levels were also observed in these patients, whereas, levels of vitamin B thiamin, biotin and pantothenic acid 12’ were not altered by peritoneal dialysis. Whitehead g£H§1., (1968) also reported a loss of folic acid during hemodialysis and was able to isolate this vitamin in the dialysate fluid. These investigators reported that folic acid loss occurred less rapidly than did urea and creatinine. Sullivan 32 21. (1970) has reported a decline in the mean plasma ascorbic acid levels of patients undergoing dialysis with the twin coil hemodialyzer. Guskin 23.31, (1970) reported oral bleeding among 29 of 70 patients studied with uremic syndrome. Ascorbic acid deficiency was 12 not implicated as a possible cause by these authors. Jones (1970) has observed patechiae and hematomas in patients under- going maintenance hemodialysis. Although blood levels of aScorbic acid were not measured, these symptoms were relieved by treatment with high doses of ascorbic acid (500-700/day). Ascorbic acid Zilva in 1925 did much of the early work on the isola- tion of L-ascorbic acid and established the general chemical prOperties of this vitamin. L-ascorbic acid (L-threo-hexono- l, 4—1actono-2-ene) is a white crystaline Solid with the chemical formula C6H8O6 and a molecular weight of 176.12g/ mole. Figure 1 illustrates the chemical configuration of the reduced (L-ascorbic acid) and the oxidized (L-dehydroascorbic acid) forms of the vitamin (Sebrell and Harris, 1967). Figure 1. Chemical configuration of L-ascOrbic acid "0 OH 0 § / 0 TR. 157: “03"" \o/ ‘0 HO -f-u \o/ §o cuzou cuzo H L-ascorbic acid L-dehydroascorbic acid Ascorbic acid is very water soluble, 1 gram dissolves in 5 m1 of water. Urea is three times as soluble as ascorbic acid, 1 gram dissolves in 1 ml of water (Merck, 1960). Man and other primates, guinea pigs, the red vented Bulbul bird and the fruit eating bat are dependent upon an exogenous source of vitamin C (King, 1967). Recently evidence has es- tablished a requirement of this vitamin for the Rainbow trout, 13 and the Coho, Sockeyed, and Chinook salmon (S. Kitamura.§£ 31., 1965; Halver gt_§1., 1962). Suggestive evidence may also establish a requirement for many other varieties of fish (Aoe §3_21,, 1971). The characteristic features of a vitamin C de- ficiency, approximately in order of evidence, are: decreased urinary excretion, decreased plasma concentrations, decreased tissue and leucocyte concentration, weakness, lassitude, suppressed appetite and growth, anemia, height- ened risk of infection, tenderness to touch, swollen and inflamed ankle joints, shortness of breath, fevers, patechial hemorrhages from the venules, beading or fracture of ribs at costochondral junctions, x-ray "scurvy lines" of tibia or femur, fracture of epiphysis, mas- sive subcutaneous, joint, muscle, and intesti- nal hemorrhages. The major physical changes observed in scurvy are asso— ciated with the failure to maintain collagen. Robertson (1961) reported that maintenance of preformed collagen does not generally require ascorbic acid. Small amounts of col- lagen are formed in the absence of ascorbic acid, however, rapid synthesis of this tissue requires ascorbic acid. Gould (1961) has shown that "growth" collagen is essentially ascorbic acid independent whereas "repair" collagen is as- corbic acid dependent. It has been fairly well established (Gould, 1961, Robertson, 1961) that ascorbic acid acts in the conversion of proline to hydroxyproline, a major amino acid constituent of collagen which must be hydroxylated after it is built into the polypeptide chain (Lehninger, 1970). Ascorbic acid has been associated with the release of free folic acid from folic acid conjugates in food (Vinter, 1968), and Nicol 23,51., (1950) have shown that ascorbic acid 14 is involved in the metabolism of folic acid to its active form, citrovorum factor. Mazur (1961) has reported that ascorbic acid and adenosine triphOSphate are involved with the biochemical mechanism for the transfer of plasma-bound iron to the liver and its subsequent incorporation into ferritin. McCurdy and Dern (1968) reported that ascorbic acid potentiates the absorption of iron from the intestinal tract. The potentiation increases with increasing doses of ascorbic acid with a cutoff point at 500mg and remains true with doses of iron up to 120mg. Binding 2; Ascorbic Agid, Pal and Guda (1959) first reported evidence indicating the presence of a combined ascorbic acid "ascorbigen" in plant tissue. Ghosh and Guda (1959) further reported that this ascorbigen could be extracted from cabbage with chloro- form and could be separated from free ascorbic acid. The fact that symptoms of scurvy appear long after the plasma ascorbic acid levels have fallen to zero (Sargent, 1967) along with the observation that ascorbic acid appears in the sweat only to a slight extent (Mickelsen and Keys, 1945) may indicate that this vitamin exists in a non-diffusible form in the blood. Holtz gthgl., (1940) and Holtz and walter (1940) reported the presence of a bound form of ascorbic acid which would not appear in the protein-free filtrate of human blood. The bound ascorbic acid was liberated by acid or enzymatic hydrolysis; Holtz reported bound ascorbic acid in 15 cell free organ extracts and milk as well as blood. Wach- holder 2£.§$° (1940) were unable to find this bound ascorbic acid in blood or milk but did find considerable amounts in the heart muscle. Sargent and Golden (1950) using a dif- fusion technique failed to find a bound ascorbic acid in blood. However, further studies by this group (Golden and Sargent, 1951) revealed that ascorbic acid moves into the erythrocyte more quickly than it moves out, indicating that ascorbic acid is not transferred across the red cell mem- brane by simple diffusion and in fact ascorbic acid may be bound to the red blood cell. OBJECTIVES OF THE PRESENT STUDY The present study was designed to 1) determine the permeability of ascorbic acid in water through a semi- permeable hemodialysis membrane, 2) to study the rates of 1 transfer of ascorbic acid from plasma, and 5) to determine whether some ascorbic acid is present in the blood as a a non-diffusible complex. The high water solubility of ascorbic acid and its low molecular weight makes a high permeability constant quite likely. No report of this permeability constant was found in the literature. The second objective is important since anemias due to deficiencies in iron and folic acid have been well documented in hemodialysis patients (Comty 33.21., 1968; Hampers gt.§l., 1967). Previous workers have Shown that ascorbic acid is associated with iron and folic acid metab- olism. (Mazur, 1961; Nicol 23 31., 1950) It may be that losses in the dialysate solution contribute to these con- ditions. The fact that plasma levels of ascorbic acid seem to be independent of the levels in the sweat may indicate that this vitamin exists in the blood in a form which makes it non-diffusible at least as far as the sweat gland is con- cerned. (Mickelsen and Keys, 1945) This may also be true of the hemodialysis membrane. 16 METHODS Apparatus Inlvitro studies: A small Babb-Grimsrud (1968) parallel flow hemodialyzer was used for this study in the determination of the perme- ability of ascorbic acid and its diffusion from plasma (Figure 2). This hemodialyzer is constructed of two 6 x 6 x 1 inch lucite plates each of which has a 4 x 4 x 1/8 inch rec- tangular depression milled in its center. A 4 x 4 x 1/8 inch piece of foam nickel metal sits in the depression flush with the lucite. The density of the foam nickel is about 5% of the solid nickel (Roth, 1970). In Operation the plates are bolted together (Figure 2); blood headers, used to collect the blood, are attached to each end of the block with rubber gaskets to insure a tight fit. Stainless steel spacers were used to control the height of the blood channel. The dialysis area was approximately 200 cm2. Ninty-five percent of this area equals the effective area which was approximately 190 cm2. Two variable flow pumps were used to deliver the fluids: the dialysate pump, a Maisch constant metering pump Operated with a flow rate of approximately 800m1/min; the blood pump, a Sigma hand pump, delivered at a rate ranging from 5 to 50 ml/min depending upon the experimental design. Figure 4 illustrates the complete system used in this study. 17 l8 CuprOphane PT 150 membranes of regenerated cellulose were used throughout this study. These membranes have a dry thinkness of about 0.5x10‘3 inch, and a wet thickness of about 1.0x10'"3 inch (Roth, 1970). This is a common clinically used membrane and discriminates on the basis of size and shape, small ions and molecules permeating relatively easily, whereas increasing size decreases permeability. Distilled water was used for the dialysate Side of the permeability series of experiments. Hemotrate formula 5, (Table 1), was used as the dialysate solution for all the experiments utilizing blood plasma. This solution is iso- tonic with reSpect to blood plasma. .‘g vivo studies: Commercial Kiil hemodialyzers in a local hospital were used for the in_yiyg studies with all subjects except one where a Travenol twin coil was substituted. Kiil hemo- dialyzers are parallel flow dialyzers. A larger Babb- Grimsrud hemodialyzer was used for the in_yiyg dog studies. Hemotrate formula 5 was used in the experiments with the human subjects. Isotonic Ringer!s and potassium free Ringer's solutions were used in the canine experiments. Ascorbic Acid and Potassium Chloride Solutions Solutions of 0.176mg/ml and 0.01mg/m1 of L-ascorbic acid in distilled water were used on the blood side for the determinations of permeability. Originally 0.176mg/ml ascorbic acid was used to decrease the possibility of any 1m 19 losses of ascorbic acid due to oxidation in air. Concen- trations of 0.01mg/ml, a concentration nearer to the physiolog- ical level in human blood, were then studied to determine if a 10 fold concentration difference would create a difference in the permeability of this membrane to ascorbic acid. The permeability of ascorbic acid was determined by pushing a distilled water solution containing ascorbic acid through the blood side of the dialyzer at varying flow rates (7-22m1/min). This solution was dialyzed against distilled water delivered at a rate of 800m1/min, a rate great enough to reduce film resistance on the dialysate side to zero. The ascorbic acid was kept well mixed by an automatic stirrer. A closed 100p system (Figure 4) was used. This meant that the ascorbic acid solution was returned from the dialyzer directly to the container from which it originally went to the dialyzer. There was, therefore, an uninterrupted flow of solu- tion through the system at all times. A 5m1 sample was taken from the inlet and outlet ports every three minutes in each run. Usually four sets of samples were taken each trial. Ascorbic acid was determined by the method of Roe and Kuether (1958) immediately after the trial was completed. A total of 8 trials were run. The permeability of KCl through this membrane was deter- mined using a solution of 20 mEq KCL in distilled water. Measurements of the KCl concentration of this solution was taken every 15 minutes. Exponential decay of the concentra- tion of the KCL solution in this system was determined by a conductivity' bridge. Asses .sec my new a Hwfivoaom Ho 3 o; sescaa cam am ommbm Each 4mm 0H2 mmommm mzammzmz .N onsmam amo m mammMA4HQ mmm