Serum Retinol, Retinol-Binding Protein, and Transthyretin in Children Receiving Dialysis

Serum Retinol, Retinol-Binding Protein, and Transthyretin in Children Receiving Dialysis Nancy Fassinger, PhD, RD,*† Abubakr Imam, MD,† and David M. Klurfeld, PhD* Objective: We investigated the relationships of retinol (ROH), retinol-binding protein (RBP), and transthyretin (TTR) in children with end-stage renal disease (ESRD). Our hypothesis was that levels of ROH and RBP would be elevated in children with ESRD. Methods and Patients: We measured ROH, RBP, and TTR serum concentrations in a group of pediatric ESRD patients biannually. Children were grouped according to age and method of dialysis, i.e., hemodialysis (HD) or peritoneal dialysis (PD): HD1, aged ,12 years (n 5 8); PD1, aged ,12 years (n 5 19); HD2, aged $12 years (n 519); and PD2, aged $12 years (n 5 29). Results: No differences in ROH, RBP, TTR, or their ratios were found as a function of type of dialysis in groups PD2 and HD2. The ROH and TTR were significantly higher in PD1 than HD1 (P 5 .01 and P 5 .003, respectively). No correlations were evident between ROH and RBP or TTR with length of time on dialysis, serum calcium, or serum creatinine, except for group PD2, in which ROH was positively correlated with RBP (P 5 .025). There were no significant differences among any of the ratios in terms of age or method of dialysis. Conclusions: The data indicate that children with ESRD exhibit elevated levels of serum ROH, RBP, and TTR, in proportions similar to those reported in the adult ESRD literature. Further study is needed to clarify the consequences of increased ROH in uremic children. Ó 2010 by the National Kidney Foundation, Inc. All rights reserved

I

T IS ESTABLISHED that adults with end-stage renal disease (ESRD) exhibit high levels of serum retinol (ROH), retinol-binding protein (RBP), and transthyretin (TTR),1 but these are not routinely measured as part of standard ESRD treatment protocols. Several studies reported that ROH and RBP were consistently elevated in adult ESRD patients, with serum concentrations of ROH and RBP at 3.5 to 5 times normal but with normal or low molar ratios of ROH to RBP.2–5 In view of these findings, it is assumed that increases in ROH and RBP are not harmful, but are a result of the diseased kidney’s inability to degrade and excrete RBP.6 Few studies

*Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan. †Children’s Hospital of Michigan and Department of Pediatrics, Wayne State University, Detroit, Michigan. A.I. is presently at the Children’s Hospital Medical Center of Akron, Akron, Ohio. D.M.K. is presently at the Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland. Address reprint requests to Nancy Fassinger, PhD, RD, Children’s Hospital of Michigan, 3901 Beaubien, Detroit, MI 48201. E-mail: [email protected] Ó 2010 by the National Kidney Foundation, Inc. All rights reserved 1051-2276/10/2001-0000$36.00/0 doi:10.1053/j.jrn.2009.05.005

Journal of Renal Nutrition, Vol 20, No 1 (January), 2010: pp 17–22

reported on serum concentrations of ROH, RBP, and TTR in children with chronic kidney disease. The limited literature for the pediatric age group presents conflicting data.7,8 Our investigation began after a 12-year-old girl with ESRD, who had never been on human growth hormone, presented at our nephrology service with pseudotumor cerebri. Her level of ROH was 8.2 mmol/L (normal, 0.9 to 2.4 mmol/L), her level of RBP was 22.2 mg/dL (normal, 3 to 6 mg/dL), and her level of TTR was 52.2 mg/dL (normal, 17 to 42 mg/dL). There is no consensus on the presumed safe levels of ROH and RBP for the pediatric ESRD population, nor are clinical signs of toxicity easily distinguishable from those of uremia before an onset of pseudotumor cerebri. The ROH and RBP of all dialysis patients at our center are measured and recorded as part of routine laboratory assessment protocols. We tested the hypothesis that ROH and RBP would be high in children with ESRD, and that the ratio of ROH to RBP would be similar to that found in adults with ESRD.

Patients and Methods To determine if serum ROH and RBP levels in children with ESRD were similar to those of 17

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FASSINGER ET AL

adults with ESRD, a retrospective chart review of ROH, RBP, and TTR levels was conducted. This study was reviewed and approved by the Human Investigation Committee of Wayne State University. Seventy-five sets of laboratory data from pediatric dialysis patients were reviewed. Transthyretin was already part of the nutrition assessment protocol, as were other standard monthly measurements such as blood urea nitrogen, serum creatinine, serum electrolytes, calcium, phosphorus, alkaline phosphatase, and triglycerides. Patients were divided into two groups according to age, and then again by method of dialysis. No patient was receiving a vitamin supplement with vitamin A. However, vitamin A intake was not quantified. Separation by method of dialysis was performed to identify differences, if any, in vitamin A status between those on hemodialysis (HD) and those on peritoneal dialysis (PD). Age separation was necessary because normal ROH levels for children aged ,12 years are quite different from those for children aged $12 years (0.7 to 1.7 mmol/L vs. 1.0 to 3.2 mmol/L, respectively).9 Groups of children included those on HD, aged ,12 years (HD1, n 5 8); those on PD, aged ,12 years (PD1, n 5 19); those on hemodialysis, aged 12 to 18 years (HD2, n 5 19); and those on peritoneal dialysis, aged 12 to 18 years (PD2, n 5 29). Because children with renal disease exhibit abnormal growth and often fall below the 5th percentile for age on the National Center for Health Statistics growth charts, and because available comparison data for ROH are based on body surface area before the onset of puberty, three children with a body surface area ,5th percentile for 12-year-olds, who were assessed at Tanner stage 1,10 were grouped with the youngest patients, although they were above age 12 years. In the PD1 group, one child received growth hormone, two received prednisone, and 14 of the 19 received nighttime tube-feeding to provide approximately 75% of their individual nutrient requirements. No subjects in the HD1 group received growth hormone, four received prednisone, two received anticonvulsants, and 3 of 8 received nighttime tube-feeding for approximately 75% of their estimated nutrient requirements. Of the PD2 subjects, none received growth hormone, two received prednisone, nine received anticonvulsants, four received 50% of their estimated nutrient requirements via

nighttime tube-feeding, and two received occasional oral supplements. No HD2 subjects received growth hormone, one received prednisone, two required anticonvulsants, one received 50% of his estimated nutrients via gastrostomy tube (GT), and two received occasional oral supplements. The HD patients fasted. Peritoneal dialysis patients had indwelling peritoneal fluid containing glucose, and were not considered to be fasting. Blood was drawn, protected from light, and immediately transported to an outside laboratory for analysis of retinol by high-pressure liquid chromatography (HPLC) with ultraviolet (UV) detection. The RBP was measured by nephelometry at the same laboratory. The TTR was analyzed by the Detroit Medical Center’s laboratory, using turbidimetry. Both laboratories are certified by the Office of the Inspector General for the Center for Medicaid and Medicare Services, and perform these assays on a routine basis. Data were analyzed with Student’s t-test, analysis of variance, and regression analysis, as applicable, using Statistix 4.1 (Analytical Software, Tallahassee, FL).

Results Comparisons of groups based on method of dialysis showed no significant differences in ROH, RBP, and TTR between the groups PD2 and HD2 (Table 1). However, serum ROH concentrations in group PD1 were significantly higher than those in HD1 (P 5 .01). There was no difference between groups for RBP. The TTR was significantly higher in the PD1 group than in the HD1 group (P 5 .01). Because triglycerides (TGs) affect the serum concentrations of some fat-soluble vitamins, we compared the TGs of HD and PD in the respective age groups. There was a significant difference in TG levels between PD1 and HD1, and TG levels in the PD1 group were higher than in the HD1 group (P 5 .01). There was no significant difference in TGs between PD2 and HD2, and no correlation between ROH and TGs within any groups (data not shown). We found no statistically significant correlations between ROH, RBP, or TTR with length of time on dialysis, serum calcium, or serum creatinine, except for group PD2, in which ROH and RBP were positively correlated (r 5 0.75, P 5 .025). The ratios of ROH to the two binding proteins

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ROH, RBT, AND TTR IN CHILDREN ON DIALYSIS Table 1. Comparisons of ROH, RBP, and TTR According to Method of Dialysis

PD1 (n 5 19) HD1 (n 5 8) P value PD2 (n 5 29) HD2 (n 5 21) P value

ROH (mmol/L)

RBP (mmol/L)

TTR (mmol/L)

TG (mg/dL)

4.6 6 1.8 3.4 6 0.6 .01 4.4 6 1.6 3.6 6 1.4 .09

9.3 6 2.2 6.6 6 1.1 .20 8.0 6 2.4 7.3 6 1.8 .36

6.6 6 1.7 5.1 6 0.6 .003 7.1 6 3.6 6.1 6 1.4 .48

268 6 117 171 6 39 .004 225 6 140 166 6 128 .21

Values are mean 6 SD. ROH, serum retinol; RBP, retinol-binding protein; TTR, transthyretin; TG, triglycerides; PD1, children aged 0 to 11 years on peritoneal dialysis; HD1, children aged 0 to 11 years on hemodialysis; PD2, children aged 12 to 18 years on peritoneal dialysis; HD2, children aged 12 to 18 years on hemodialysis.

and the ratio of the binding proteins to each other were calculated. There were no significant differences between any of these ratios in terms of age or method of dialysis (Table 2).

Discussion This study established that measured levels of ROH, RBP, and TTR in children with ESRD are similar to those reported earlier for adult patients with ESRD (Table 3). These values for serum ROH concentrations are also in accordance with those in the study of Kriley and Warady7 in a group of children receiving PD. Conversely, a study conducted in Poland with children receiving HD reported lower-than-normal values for serum ROH when compared with control subjects, although both patients and subjects in that study had serum ROH values within normal limits (0.7 to 2.6 mmol/L).8 Levels of RBP and TTR were not reported in either study.7,8 Typically, the ROH:RBP ratios reported in ESRD adult studies range from 0.40 to 0.73. (Table 4).11,12 The ratio of ROH:RBP in our study group was 0.74. Method of dialysis did not appear to influence any of the values measured. When ROH, RBP,

and TTR were compared in terms of the two methods of dialysis, the only differences were in the younger age group, in which both ROH and TTR were significantly lower in children treated with HD. Because only the two younger groups had significantly different serum concentrations of ROH, TG, and TTR when method of dialysis was compared, we reviewed other clinicalmanagement issues that may have influenced ROH, TR, and TTR. Triglycerides may be higher in children on PD because samples are drawn in the postabsorptive state, and glucose in dialysate is lipogenic. A possible explanation for the differences in ROH, TTR, and TTR between groups PD1 and HD1 could be related to the fact that a larger proportion of children in the PD1 group were fed via GT. The PD1 group had a higher proportion of patients fed .75% of their total daily calories with commercially prepared formulas: 14 of 19 (78%) of the PD1 patients versus 3 of 8 (38%) of the HD1 patients were fed via GT. The formula used for GT feeding in all patients had been manufactured for adult ESRD patients, and was diluted to a caloric density well-tolerated by the children. Each GT feeding regimen provided adequate calories for the individual patient. Caloric intake for

Table 2. Ratios of ROH to Binding Proteins According to Method of Dialysis

PD1 (n 5 19) HD1 (n 5 8) P value PD2 (n 5 29) HD2 (n 5 21) P value

ROH:RBP

ROH:TTR

RBP:TTR

0.61 6 0.26 0.54 6 0.17 .70 0.54 6 0.14 0.52 6 0.20 .62

2.14 6 0.38 2.0 6 0.19 .64 1.77 6 0.26 1.80 6 0.27 .72

1.94 6 0.33 1.95 6 0.22 .71 1.98 6 0.98 1.85 6 0.29 .62

Values are mean 6 SD. ROH, serum retinol; RBP, retinol-binding protein; TTR, transthyretin; PD1, children aged 0 to 11 years on peritoneal dialysis; HD1, children aged 0 to 11 years on hemodialysis; PD2, children aged 12 to 18 years on peritoneal dialysis; HD2, children aged 12 to 18 years on hemodialysis.

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Table 3. Comparisons of Published Studies Describing Vitamin A and Its Binding Proteins in Adults With End-Stage Renal Disease Vitamin A (mmol/L) Study

MVI

[1] [3] [4] [5] [6] [10] [19]

3.57 7.2 5.2 6.6

[20] [21] [22]

3.8

[23]

No MVI

C

RBP (mmol/L) MVI

No MVI

12.9

13.1

12.5

11.8 7.9 13.6 10.3H 5.4T 13.62H 2.14T

TTR (mmol/L) C

No MVI

C

1.43 6.4 3.9 5.36 4.8 5.5 7.48H 5.71T 4.62H 4.41H 3.7 6.4ND 4.5D 7.0

1.8 1.7 1.8 3.6 2.5

9.9ND 7.3D 11.77

2.16

2.7 2.1 3.0 3.19

Dialysis Method

Analysis Method F LC/UV F F F/LC F F

5.0

4.8

7.69H 5.36T 5.2H 7.2T

7.2

H P H H H H H/T

5.55

H/T

F

P P

F

H

LC

2.76

RBP, retinol-binding protein; TTR, transthyretin; MVI, multivitamin; C, control; H, hemodialysis; P, peritoneal dialysis; T, transplant; F, fluorometric method; LC, HPLC separation; UV, ultraviolet absorbance detection method; D, diabetic; ND, nondiabetic.

children in this age group ranged from 70 to 120 kcal/kg/day. The retinyl palmitate and proportionately higher fat content of commercially prepared feedings may have altered ROH, TTR, and TG in the PD1 group. The formula used was designed to be lower in vitamin A per calorie than in standard enteral feeding formulas. However, it did contain preformed vitamin A in the form of retinyl palmitate (348 mg/L, or 0.175 mg/kcal). The fat content of the formula was 43% of total calories, from a mixture of high oleic safflower and soy oils. Most children who were fed via GT received between 1000 and 1500 kcal/day or 175 to 263 mg of retinyl palmitate, and 48 to 72 g/day of polyunsaturated fat. Fewer children in the HD1 group (38%, vs. 78% in the PD1 group) were fed via

GT. This may account for the higher ROH concentrations in the PD1 group. Formula-fed patients’ diets may also contain significantly more vitamin A than the diets of those who do not receive GT feeding or other supplements, because diets for renal patients typically tend to be relatively low in vitamin A due to restrictions on foods high in phosphorus (dairy) and potassium (deep yellow and green leafy vegetables). The only patient who exhibited overt signs of vitamin A toxicity did not receive tube-feeding, but did receive a daily multivitamin with 5000 IU of retinyl palmitate. None of the patients fed via GT exhibited overt signs of vitamin A toxicity. Although we report on elevated serum concentrations of ROH and its binding proteins in children with ESRD, we have

Table 4. Ratios of ROH to Binding Proteins According to Published Studies ROH:RBP

ROH:TTR

Study

MVI

No MVI

C

[5] [22] [10] [3] [24]

0.52

0.45 0.61 0.40 0.49 0.73H 1.0T 0.65ND 0.62D 0.59

0.63 0.86

[22] [23]

0.56

1.2

MVI

No MVI

RBP:TTR C

No MVI

C

0.96

0.38

1.5

0.44

0.97H 1.1T

0.5

1.34H 1.01T

0.42

Dialysis Method

Analysis Method

H H H P H/T

F F/LC F LC/UV F

P 0.79

H

LC

ROH, serum retinol; RBP, retinol-binding protein; TTR, transthyretin; MVI, multivitamin; C, control; H, hemodialysis; P, peritoneal dialysis; T, transplant; F, fluorometric method; LC, HPLC separation; UV, ultraviolet absorbance detection method; D, diabetic; ND, nondiabetic.

ROH, RBT, AND TTR IN CHILDREN ON DIALYSIS

not described intracellular concentrations of ROH or retinoic acids. This is an important issue that needs to be resolved, because with the exception of the visual cycle, it is the nuclear action of retinoic acids that is responsible for the actions of vitamin A. Given that ROH carrier proteins are also elevated in ESRD, an examination of the mechanism of transport of retinol across the cell membrane is important. At present, this mechanism is not completely understood. Studies indicate the membrane transport of retinol by membranebound RBP receptors, endocytosis, diffusion based on intracellular free ROH concentration gradients, and a form of ROH shuttling between extracellular and intracellular carrier proteins.13–16 Recent reports found increasing evidence for the existence of a cell-membrane receptor that binds RBP.14 The method of membrane transport for retinol is particularly important in the discussion of its metabolism in renal disease, because serum concentrations of both ROH and RBP are abnormal, and may influence intracellular concentrations of ROH and retinoic acid. Furthermore, current therapy for renal osteodystrophy may include high doses of 1,25-dihydroxyvitamin D3 that could disrupt the balance of ligands necessary for the respective nuclear proteins to regulate gene transcription, particularly those genes whose products are necessary for calcium homeostasis. Increased attention to nutrition, 1,25-dihydroxyvitamin D3 therapy, and the daily administration of growth hormone has improved the outcomes of many children in the past 15 to 20 years. However, despite these interventions, many children still experience growth failure, decreased muscle mass, pruritus, hypercalcemia, hyperlipidemia, and abnormal bone growth.17 It is assumed that RBP not catabolized and excreted by the damaged kidney acquires more retinol and returns to the plasma, increasing the total vitamin A levels of patients with ESRD.11 However, this assumption is not consistent with what is known about normal apo-RBP recycling. Animal studies suggest that when serum ROH is at normal or high levels, RBP is sequestered in the liver, and additional ROH is not added to the circulation.14 In the patient with ESRD, intracellular transport may be abnormal because serum concentrations of carrier proteins are also very high. If, in fact, transmembrane movement is dependent on concentration gradients of ROH or its carrier proteins, the equilibrium constants could be distorted,

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causing either increased or decreased intracellular concentrations of ROH. Further complicating the picture is the inability to determine clinically the impact of elevated serum ROH in the patient with ESRD, because the symptoms of uremia, vitamin A toxicity, and vitamin A deficiency are similar. Current recommendations discourage the use of multivitamins containing fat-soluble vitamins in all patients with ESRD, including children.18 In addition, vitamin A intake may be indirectly limited by dietary restrictions imposed to maintain acceptable serum potassium and phosphorus levels in the face of decreased renal clearance of these minerals. Given the rapid cellular turnover and growth of children, the maintenance of intracellular retinoic acid levels is very important. However, in light of high total vitamin A levels, and without an understanding of the intracellular availability of ROH and retinoic acid, supplementation with vitamin A is risky. Because the action of vitamin A is hormone-like and its actions are closely tied to other growth-related hormones, it is important to identify abnormalities in its metabolism in the uremic state, especially those abnormalities in children with renal failure.

Conclusions The data reported here indicate that children with ESRD have elevated levels of serum ROH, RBP, and TTR, in proportion to those levels reported in the early adult ESRD literature. This anomaly deserves further investigation, given the plethora of new knowledge concerning the action of vitamin A and its metabolites that has been elucidated since the reported findings of increased ROH and RBP in adult ESRD patients. Most importantly, the intracellular actions of retinoic acids in the uremic state should be investigated.

References 1. Werb R, Clark WF, Lindsay RM, et al: Serum vitamin A levels and associated abnormalities in patients on regular dialysis treatment. Clin Nephrol 12:63-68, 1979 2. Blumberg A, Hanck A, Sander G: Vitamin nutrition in patients on continuous ambulatory peritoneal dialysis (CAPD). Clin Nephrol 20:244-250, 1983 3. Farrington K, Miller P, Varghese Z, et al: Vitamin A toxicity and hypercalcaemia in chronic renal failure. Br Med J [Clin Res] 282:1999-2002, 1981 4. Stewart WK, Fleming LW: Plasma retinol and retinol binding protein concentrations in patients on maintenance

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haemodialysis with and without vitamin A supplements. Nephron 30:15-21, 1982 5. Vahlquist A, Berne B, Berne C: Skin content and plasma transport of vitamin A and beta-carotene in chronic renal failure. Eur J Clin Invest 12:63-67, 1982 6. Cundy T, Earnshaw M, Heynen G, et al: Vitamin A and hyperparathyroid bone disease in uremia. Am J Clin Nutr 38: 914-920, 1983 7. Kriley M, Warady BA: Vitamin status of pediatric patients receiving long-term peritoneal dialysis. Am J Clin Nutr 53: 1476-1479, 1991 8. Zwolinska D, Grzeszczak W, Szczepanska M, et al: Lipid peroxidation and antioxidant enzymes in children on maintenance dialysis. Pediatr Nephrol 21:705-710, 2006 9. Pilch S: Analysis of vitamin A data from the health and nutrition examination surveys. J Nutr 117:636-640, 1987 10. Marshall WA, Tanner JM: Variations in pattern of pubertal changes in girls. Arch Dis Child 44:291-303, 1969 11. Kelleher J, Humphrey CS, Homer D, et al: Vitamin A and its transport proteins in patients with chronic renal failure receiving maintenance haemodialysis and after renal transplantation. Clin Sci 65:619-626, 1983 12. Stein G, Schone S, Geinitz D, et al: No tissue level abnormality of vitamin A concentration despite elevated serum vitamin A of uremic patients. Clin Nephrol 25:87-93, 1986 13. Dong D, Ruuska SE, Levinthal DJ, et al: Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid. J Biol Chem 274:23695-23698, 1999 14. Noy N: Retinoid-binding proteins: mediators of retinoid action. Biochem J 348:481-495, 2000 15. Sundaram M, Sivaprasadarao A, DeSousa MM, et al: The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor. J Biol Chem 273:3336-3342, 1998

16. Vieira AV, Schneider WJ, Vieira PM: Retinoids: transport, metabolism, and mechanisms of action. J Endocrinol 146: 201-207, 1995 17. Rashid R, Neill E, Maxwell H, et al: Growth and body composition in children with chronic kidney disease. Br J Nutr 97:232-238, 2007 18. National Kidney Foundation-Dialysis Outcomes Quality Initiative: NKF-DOQI clinical practice guidelines for the treatment of anemia of chronic renal failure. Am J Kidney Dis 30(Suppl):S192-S240, 1997 19. Kelleher J, Humphrey CS, Homer D, Davison AM, Giles GR, Losowsky MS: Vitamin A and its transport proteins in patients with chronic renal failure receiving maintenance haemodialysis and after renal transplantation. Clin Sci (Lond) 65: 619-626, 1983 20. Stein G, Schone S, Geinitz D, et al: No tissue level abnormality of vitamin A concentration despite elevated serum vitamin A of uremic patients. Clin Nephrol 25:87-93, 1986 21. Mydlik M, Derzsiova K, Valek A, Szabo T, Dandar V, Takac M: Vitamins and continuous ambulatory peritoneal dialysis (CAPD). Int Urol Nephrol 17:281-286, 1985 22. Vahlquist A, Berne B, Danielson BG, Grefberg N, Berne C: Vitamin A losses during continuous ambulatory peritoneal dialysis. Nephron 41:179-183, 1985 23. Delacoux E, Evstigneeff T, Leclercq M, et al: Skin disorders and vitamin A metabolism disturbances in chronic dialysis patients: the role of zinc, retinol-binding protein, retinol and retinoic acid. Clin Chim Acta 137:283-289, 1984 24. Vannucchi MT, Vannucchi H, Humphreys M: Serum levels of vitamin A and retinol binding protein in chronic renal patients treated by continuous ambulatorial peritoneal dialysis. Int J Vitam Nutr Res 62:107-112, 1992