Plasma Retinol-binding Protein Levels Research Articles

P203 PLASMA TRANSTHYRETIN FORMS IN PATIENTS WITH WILD–TYPE TRANSTHYRETIN CARDIAC AMYLOIDOSIS: INFLUENCED OF TAFAMIDIS TREATMENT

Abstract Introduction Transthyretin (TTR) is a plasma protein that transports thyroxin and retinol complexed to retinol–binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid in the myocardium, causing TTR cardiac amyloidosis (ATTR–CA). Aim of this study is to develop a native electrophoretic method to characterize circulating TTR forms in plasma samples of ATTR–CA patients, before and after administration of tafamidis, a TTR stabilizer. Material & Methods Plasma samples from patients affected by wild–type ATTR amyloidosis (ATTRwt, n=10) were collected before (T0) and during tafamidis treatment at 14, 30 and 90 days. Plasma samples of sex– and age–matched healthy controls (n=6) were also collected. All samples were separated on a native 4–20% Tris–Gly polyacrylamide gel. Western blot analysis was performed with anti–TTR or anti–RBP antibodies. Proteins were detected by Clarity ECL substrate. ImageLab software was used for image acquisition and densitometric analysis of the blots. Total TTR (mg/dl) and RBP (mg/dl) were quantified by nephelometry. Results In both groups, the most represented forms were: TTR dimers or trimers (37–50kDa), TTR tetramers complexed with RBP protein in 1:1 (75kDa,) or 1:2 ratio (100kDa), and high molecular weight aggregates (>150kDa). RBP protein was detectable in association with TTR tetramers and some of the TTR aggregates (250kDa, 480kDa). Based on TTR electrophoretic pattern at T0, patients could be divided in 2 groups: ATTR–1, characterised by the presence of TTR tetramers (55kDa), and ATTR–2, characterised by its absence. The ATTR–1 group showed also higher levels of total TTR in comparison with ATTR–2 (mean±SD 25.5±3.8, 16.6±4.2 respectively, p=0.004), while plasma RBP levels were similar (5.1±0.7, 5.1±1.4 respectively). The electrophoretic patterns of ATTR–1 at T0, as well as TTR and RBP levels were comparable to those of controls (TTR 22.6±3.8, RBP 4.4±0.9). Tafamidis treatment was associated with a progressive increase of total TTR levels both in ATTR–1 and –2 (T90 31.4±5.8, 28.7±6.8) but the 2 groups continued to be distinguishable by the electrophoretic pattern. Conclusions The native electrophoresis allowed us to detect diverse circulating TTR forms and to classify patients in 2 groups according to their electrophoretic pattern. TTR quali/quantitative evaluation by electrophoresis could represent a valid tool to assess the individual pharmacological response to tafamidis.

Assessment of Vitamin A Status in Patients with Iron Deficiency Anemia

Introduction: Iron and vitamin A deficiency are two very prevalent and easily preventable nutrient deficiencies. This study was conducted to assess vitamin A status in patients with iron deficiency anemia and to further study the correlation of vitamin A status with biochemical markers of iron deficiency. Materials and Method: Eighty patients with iron deficiency anemia were enrolled and investigated for a complete blood count, an iron profile, liver, and kidney function tests and plasma retinol binding protein levels. The mean age of patients was 31.14 ± 11.33 years, with a range of 16 to 62 years. Results: Mean hemoglobin was 7.19 ± 2.1 g/dL. Serum iron, ferritin, and transferrin saturation were low in all patients, while total iron binding capacity (TIBC) was elevated in only 74 patients (94.81%). Nineteen patients (23.8%) had vitamin A deficiency, with a mean retinol binding protein (RBP) 0.53 ± 0.13 µmol/L. Vitamin A deficient patients had a mean hemoglobin of 6.8±2.14 gm/dL, mean corpuscular volume (MCV) 71.35 ± 8.86 fL, a mean corpuscular hemoglobin (MCH) 19.43±4.36 pg, mean corpuscular hemoglobin concentration (MCHC) 25.44±4.92 gm/dL, serum iron of 28.21 ± 9.73 mcg/dL, serum ferritin 13.04 ± 12.41 ng/mL, transferrin saturation 6.81 ± 3.07%, and TIBC 427.85 ± 78.57 mcg/dL. Among vitamin A deficient patients, RBP had positive correlation with serum iron and transferrin saturation; while, simultaneously showing negative correlation with serum ferritin and TIBC. Conclusion: Vitamin A deficiency affects iron metabolism, causing abnormal iron trapping and systemic iron deficiency, thus worsening the clinical profile of iron deficiency anemia. This study guides us to screen iron deficiency anemia patients for the concomitant vitamin A deficiency for efficient treatment of such patients.

An ELISA for plasma retinol-binding protein using monoclonal and polyclonal antibodies: Plasma variation in normal and insulin resistant subjects

Objectives: Plasma retinol-binding protein (RBP) has been linked to insulin resistance and cardiovascular risk, yet little is know of its natural variation in plasma. We examined this in normal subjects and compared plasma levels and variability in lean subjects and subjects with the metabolic syndrome. Methods: We established an “in house” ELISA for plasma RBP and measured levels in 20 normal subjects over daylight hours and 2 subject groups, either lean or classified with the metabolic syndrome. Results: Plasma RBP in normal subjects did not vary over the day with no differences between males and females. There was also no difference in plasma RBP levels and between the age- and sex-matched lean subjects compared to the metabolic syndrome group. Conclusion: The lack of variation in plasma RBP in normal subjects and the lack of difference between plasma RBP in normal and metabolic syndrome subjects suggest the link between plasma RBP and insulin resistance is tenuous. Investigating a large cohort over the diabetic non-diabetic spectrum may clarify this issue.

Polybrominated diphenyl ether (PBDE)-induced alterations in vitamin A and thyroid hormone concentrations in the rat during lactation and early postnatal development

In experimental animals fed standard laboratory diets, penta-BDE mixtures can decrease circulating thyroid hormone and liver vitamin A concentrations. A substantial number of pregnant women and their children have marginal vitamin A status, potentially increasing their risk of adverse effects to penta-BDE exposure. The current study investigated the effects of maternal gestational and lactational penta-BDE exposure on thyroid hormone and vitamin A homeostasis in rats of sufficient vitamin A (VAS) or marginal vitamin A (VAM) status and their offspring. Dams were administered daily oral doses of 18 mg/kg DE-71 (a penta-BDE mixture) or a corn oil vehicle from gestation day 6 through lactation day (LD) 18. Thyroid hormone and vitamin A homeostasis were assessed in plasma and tissues of LD 19 dams and postnatal day (PND) 12, 18, and 31 pups. DE-71 exposure induced hepatomegaly in VAS and VAM pups at all timepoints and increased testes weights at PND 31. While liver vitamin A concentrations were low in DE-71 treated dams and pups, plasma retinol concentrations and plasma retinol binding protein levels were only low in VAM animals exposed to DE-71. DE-71 exposure lowered plasma thyroxine concentrations in VAS and VAM dams and pups. Plasma thyroid stimulating hormone concentrations were high in VAM dams exposed to DE-71, suggesting that marginal vitamin A status enhances the susceptibility to thyroid hormone axis disruption by DE-71. These results support the concept that marginal vitamin A status in pregnant women may increase the risk for PBDE-induced disruptions in vitamin A and thyroid hormone homeostasis.

USEFULNESS OF TRANSTHYRETIN (TTR) AND RETINOL BINDING PROTEIN (RBP) DETERMINATION IN PATIENTS HIV+/AIDS

Nutritional disorders are presented in patients HIV+/AIDS. We analyze the levels of specific plasma fractions of potential utility in the assessment of nutritional status. Transthyretin (TTR) and Retinol Binding Protein (RBP) plasma levels, were determined in 55 patients between 26–44 years old (29 HIV+ and 26 AIDS), with antiretroviral treatment (HAART), by quantitative radial inmunodiffussion on plates (The Binding Site Limited, UK). Results(mg/dL) expressed as X+ SD were : TTR : HIV+: :34.1±8.9; AIDS :29.5±8.9; RBP: HIV+: 8.0± 2.6; AIDS: 7.9±1.6. No statistical differences, in both specific fractions, were observed between groups. Results of whole population (HIV+/AIDS) were: TTR:31.9±9.1, RBP:7.9±2.1. The 16.4% of patients shows TTR levels lower than 20 mg/dL, values related to protein deficiency status; the 20% of population presents values higher than reference range (20–40 mg/dL). The 45,5 % of the group with CD4<200 cel/mL presents the lowest values of TTR. It proves the compromised nutritional status, simultaneously with the infection. Moreover, the 50.9% shows plasma RBP levels higher than reference values (4–8 mg/dL). These short-lived proteins are very promissory in the evaluation of the nutritional status; nevertheless, in the studied patients, metabolic disorders caused by stress, would increase its concentration. By the other hand, the results point out the importance to incorporate the determination of TTR in the periodic nutritional control of these patients. Supported by UBA(B060)

Saturation of retinol-binding protein correlates closely to the severity of alcohol-induced liver disease

Impaired metabolism of retinol has been shown to occur in alcohol-induced liver disease (ALD). The purpose of the present study was to investigate the saturation of retinol-binding protein (RBP) in 6 patients with different stages of ALD. Hospitalized alcohol consumers ( n = 118) with different stages of ALD (ALD1: mild stage of liver damage; ALD2: moderately severe changes of the liver with signs of hepatic inflammation; ALD3: severely impaired liver function) and 45 healthy control subjects were nutritionally assessed, and retinol and RBP content was measured in plasma by high-performance liquid chromatography and enzyme-linked immunosorbent assay methods, respectively. No differences were noted in daily retinol intake, but subjects with ALD had significantly lower concentrations of retinol in plasma (ALD1: 1.81 ± 0.17 μmol/l [mean ± S.E.M.]; ALD2: 1.95 ± 0.24 μmol/l; ALD3: 0.67 ± 0.13 μmol/l) compared to controls (2.76 ± 0.19 μmol/l). Subjects of group ALD2 had significantly higher plasma RBP levels than controls ( P < .05) and patients with ALD1 ( P < .05) and ALD3 ( P < .001). The relative saturation of RBP with retinol decreased with severity of ALD (controls: 76.8 ± 5.0%; ALD1: 55.8 ± 6.5%; ALD2: 43.5 ± 6.2%; ALD3: 29.0 ± 5.1%). The present study indicates that plasma concentrations of retinol and RBP per se do not correlate to severity of ALD, but rather that the retinol/RBP ratio links to the severity of alcohol-induced liver damage. From these results, a reduced availability of retinol in the periphery due to an altered saturation of RBP can be concluded.

Retinol binding protein status in relation to ocular surface changes in patients with cystic fibrosis treated with daily vitamin A supplements

Cystic fibrosis (CF) is an autosomal recessive disease characterised by increased viscosity of mucus secretions and high chloride concentration in exocrine secretions. Clinically, the patients suffer from chronic pulmonary changes, chronic pancreatic deficiency, and an obstruction of the gastrointestinal tract. The disease affects all secretory epithelia including the eye. The influence of nutritional status on long-term survival and quality of life of CF patients is well documented. Steatorrhea, a consequence of decreased fat digestion and absorption may be associated with vitamin deficiences, including vitamin A. The aim of this study was to document plasma retinol binding protein (RBP) status, a specific plasma transport protein for vitamin A, and ocular surface changes in children and adolescents with CF. The patients were recruited at the 3rd Department of Paediatric Diseases, Medical University of Bialystok, Poland. All patients were regularly seen by a CF specialist dietitian. A group of 15 patients had the following investigations: plasma RBP, visual acuity, physical examination, tear film break-up time, fluorescein staining and Schirmer tear test. A group of 15 age- and sex-matched controls without CF or ocular pathology were also recruited. Plasma RBP concentrations were significantly lower in patients with CF than in the control group. CF patients showed a statistically significant increase in the incidence of clinical blepharitis. Five of the CF patients had clinical evidence of dry eyes. Low plasma retinol binding protein levels frequently occur in clinically stable and retinol supplemented cystic fibrosis patients, of whom five had dry eyes. We recommend monitoring of plasma retinol binding protein levels and evaluation of ocular surface changes, especially those with dry eye symptoms in all cystic fibrosis patients.

Plasma retinol binding protein levels in individuals with transthyretin gene mutations

Plasma retinol binding protein levels in individuals with transthyretin gene mutations

Relationship of holo-free and transthyretin-bound plasma retinol-binding protein levels with liver vitamin A in rats

Vitamin A containing (holo) free and transthyretin-bound (TTR) retinol binding protein (RBP) concentrations in plasma from rats with variable vitamin A status were determined by high performance liquid chromatography. Two different methods were used: (1) molecular exclusion with a TSK 2000 column and (2) reverse phase using a Protesil Octyl 300 column. Holo TTR-RBP peak areas were positively correlated to liver vitamin A concentrations ( r = 0.79 for molecular exclusion 0.81 for reverse phase) in rats with marginal and normal vitamin A status. This correlation was higher than the correlation of serum retinol to liver vitamin A in these rats ( r = 0.58). The correlation of holo-TTR-RBP to liver vitamin A was also higher than its correlations to plasma vitamin A. Therefore, plasma concentrations of holo—TTR-RBP may be influenced by marginal vitamin A liver stores to a greater extent than plasma retinol is. This suggests that holo—TTR-RBP protein concentrations may be the better indicator of marginal vitamin A nutritional status in rats. The correlations of both holo—TTR-RBP and serum retinol decreased sharply at high liver vitamin A concentrations. Neither method is suitable for measuring sub-toxicity in rats.

Pharmacokinetics of n‐4‐hydroxyphenyl‐retinamide and the effect of its oral administration on plasma retinol concentrations in cancer patients

Concurrent with a phase-II trial of 4HPR in patients with various cancers, we studied the plasma pharmacokinetics of both 4HPR and its major metabolite 4MPR as well as the effect of 4HPR administration on plasma retinol concentrations using a simple, specific and sensitive HPLC procedure. Initial estimates of plasma pharmacokinetic parameters after oral administration of 4HPR (300 mg/day) [corrected] in 3 cancer patients were the following: 4HPR, t beta 1/2 = 13.7 hr, AUC = 3.49 micrograms.hr/ml, CL = 56.57 L/hr/m2; 4MPR, t beta 1/2 = 23.0 hr, AUC = 1.15 micrograms.hr/ml, CL = 239.29 L/hr/m2. We also found that oral administration of 4HPR resulted in a rapid, profound and significant reduction in plasma retinol concentrations. The mean plasma retinol concentrations for 9 patients decreased 60% from baseline to below 200 ng/ml within 1-2 weeks of 4HPR dosing initiation. In addition, there was a concurrent, significant reduction in plasma retinol-binding protein levels in these patients. The mechanism whereby 4HPR reduces plasma retinol levels in vivo has not been determined. The addition of 4HPR to pooled human plasma at 37 degrees C in vitro did not reduce endogenous retinol levels, suggesting no direct chemical interaction between these 2 retinoids.

Significance of plasma retinol binding protein levels in recurrence of breast tumors in women.

Plasma retinol-binding protein (RBP), prealbumin, vitamin A (total) and beta-carotene levels were studied in premenopausal women with node-positive breast carcinoma receiving adjuvant chemotherapy. Plasma levels were measured prior to chemotherapy and at monthly intervals during the chemotherapy course. The results showed that significantly lower RBP levels during the course of the study were associated with early tumor recurrence. Patients who maintained a disease-free status for 24 months or longer had significantly higher plasma RBP levels than those who had tumor metastasis at distant sites within 24 months after beginning chemotherapy. RBP levels were not associated with adjuvant chemotherapy (CMF)-induced hormonal changes (amenorrhea vs. no amenorrhea), or family history of breast cancer. In contrast, breast cancer patients with a prior history of benign breast disease had significantly lower RBP levels than did healthy, premenopausal women. Reduced RBP levels in these patients are due to neither an inadequate dietary intake of beta-carotene, nor to severe protein malnutrition.

Minerals in plasma and urine and gustatory function tests for salt in a group of healthy young men.

Detection threshold for salt (NaCl) and discrimination between two levels of NaCl concentration (0.6 and 0.7%) in foods, and their relation to some selected biochemical parameters in plasma and urine (Zn, Na, K, Mg, Ca, Se for plasma and urine, Cu and retinol binding protein for plasma) were investigated in 15 healthy male college students. No subject failed to discriminate the NaCl concentrations in more than 50% of the tests. The rate of correct discrimination (RCD) was not associated with plasma Zn (P-Zn), plasma retinol binding protein (P-RBP), urinary potassium (U-K) or urinary sodium (U-Na), which significantly correlated with RCD in our previous study, while the detection threshold was significantly correlated with urinary Ca-Mg ratio (U-Ca/Mg), urinary Ca (U-Ca), U-Na, and urinary Mg. In the stepwise multiple regression analysis, U-Ca/Mg, plasma Ca, plasma Na, and RCD were selected as significant independent variables. These indicate that the status of minerals such as Na, Ca, and Mg is related to the gustatory function. One possible explanation for the discrepancy between the present and previous results is the elevated P-Zn and P-RBP levels in the present subjects.

Effect of retinol status on retinol-binding protein biosynthesis rate and translatable messenger RNA level in rat liver.

Studies were conducted to explore the role of retinol in the control of the rate of synthesis of plasma retinol-binding protein (RBP) in the liver of the rat. Previous studies have shown that nutritional retinol status strongly influences RBP secretion from the liver cell. Both the in vivo relative rate of RBP synthesis and the in vitro translatable level of RBP-specific mRNA were examined in normal vitamin A, retinol-depleted, and retinol-repleted rats. The relative rate of RBP synthesis was estimated by measuring the extent of incorporation of [3H]leucine, [3H]lysine, and [3H]phenylalanine after a 12-min pulse label into immunoprecipitable RBP, relative to the incorporation of these amino acids into total liver trichloroacetic acid-precipitable protein. The level of translatable RBP-specific mRNA was quantitated in vitro by translation of rat liver poly(A+)RNA in the rabbit reticulocyte lysate protein-synthesizing system. The amount of newly synthesized RBP was determined relative to the amount of newly synthesized total protein. Both the relative rate of RBP synthesis (approximately 0.26%) and the translatable level of RBP-specific mRNA (approximately 0.14%) were found to be constant regardless of the retinol status of the rats. These results indicate that retinol, the molecule that RBP specifically binds and transports, does not appear to control the rate of synthesis of RBP or the translatable level of RBP-specific mRNA in the liver of the rat. Regulation of plasma RBP levels by retinol must be exercised at a locus beyond that of RBP synthesis.

Plasma retinol and retinol binding protein concentrations in patients on maintenance haemodialysis with and without vitamin A supplements.

Plasma retinol and retinol-binding protein (RBP) concentrations have been estimated in patients on maintenance haemodialysis over a 4-year period. For the first 2 years multivitamin supplements containing vitamin A were taken, and for the second 2 years no vitamin A supplements were given. Mean plasma retinol concentrations decreased significantly but only from 3.8 times normal to 3.1 times normal after vitamin A supplements stopped. There was no significant change in th high plasma RBP levels. Ultracentrifugation of plasma at a salt density of 1.21 showed that nearly all the retinol was associated with RBP in the high density protein fraction, as it is with normal subjects. Column chromatography confirmed that there was no increase in plasma retinyl esters in the renal failure patients, as is found in hypervitaminosis A due to drug overdosage. The high plasma retinol and RBP levels remained remarkably stable in individual patients throughout the 4-year study. The increase in plasma RBP was possibly related to residual urine output. The results are compatible with a feedback mechanism whereby the extent of the increase in plasma RBP as renal failure develops controls the consequent high plasma level of retinol.

Plasma retinol-binding protein.

Vitamin A is mobilized from liver stores and transported in plasma in the form of the lipid alcohol retinol, bound to a specific transport protein, retinol-binding protein (RBP). A great deal is known about the chemical structure, metabolism, and biological roles of RBP. RBP is a single polypeptide chain with molecular weight close to 20,000. RBP interacts strongly with plasma prealbumin, and normally circulates in plasma as a 1:1 molar RBP-prealbumin complex. Both the primary and the tertiary structure of prealbumin are known, and the primary structure of RBP has recently been reported. Much information is available about the protein-protein and protein-ligand interactions that are involved in this transport system. Many clinical studies have examined the effects of a variety of diseases on the plasma levels of RBP and prealbumin in humans. Plasma RBP levels are low in patients with liver disease and are high in patients with chronic renal disease. These findings reflect the facts that RBP is produced in the liver and mainly catabolized in the kidneys. Delivery of retinol to extra-hepatic tissues appears to involve specific cell surface receptors for RBP. Vitamin A mobilization from the liver, and delivery to peripheral tissues, is highly regulated by factors that control the rates of RBP production and secretion. Retinol deficiency specifically blocks the secretion of RBP, so that plasma RBP levels fall and liver RBP levels rise. Injection of retinol into vitamin A-deficient rats stimulates the rapid secretion of RBP from the liver into the plasma. The cellular and molecular mechanisms that mediate these phenomena are under investigation. Elucidation of these mechanisms should help define the basic mechanisms that control the mobilization, transport, and delivery of vitamin A.

Depressed plasma retinol-binding protein (RBP) in cystic fibrosis (CF)

Decreased plasma levels with normal or increased liver stores of vitamin A were reported previously in CF patients orally supplemented with water-miscible vitamin A. These findings suggested a defect in the mechanism by which vitamin A is transported from the liver in patients with CF. Plasma levels of RBP, the specific carrier protein for vitamin A, have been measured by radioimmunoassay in 69 samples from 49 CF patients and in 96 normal age-matched controls. Mean RBP levels (± SEM) in CF, 24.4 ± 1.3 μg/ml, were significantly (p < .001) lower than in normal controls, 37.3 ± 1.3 μg/ml. In normal children 14 years and under, plasma RBP and vitamin A levels showed a signification (p < .001) correlation with age; in contrast no age-related rise was observed in CF children of comparable age. Saturation of RBO with vitamin A was less than normal (p < .004), and the levels of plasma prealbumin (the protein normally complexed with RBP) were decreased (p < .001) in the CF group 15 years and older compared with controls. Depressed plasma RBP levels were not correlated with plasma G.O.T. or albumin levels. These results suggest that a defect in the hepatic production and/or secretion of RBP occurs in CF.

Radioimmunoassay of human plasma retinol-binding protein.

A radioimmunoassay for human plasma retinol-binding protein (RBP) has been developed utilizing a double antibody precipitation technique. RBP was purified 1500- to 2000-fold by procedures described previously. A specific anti-human RBP antiserum was prepared in rabbits by three once-weekly injections of purified RBP emulsified with Freund's adjuvant. RBP was iodinated with (131)I and the RBP-(131)I was purified by gel filtration on Sephadex G-100 after complex formation with human plasma prealbumin. The RBP-(131)I was completely (> 95%) immunoprecipitable in the presence of an excess of specific antiserum, it was not (< 5%) immunoprecipitable in the absence of specific antiserum, and it could be completely displaced from antibody by excess unlabeled RBP. The standard curve obtained in the immunoassay with normal plasma was identical to that with pure RBP. Duplicate samples differed from their mean by 5 +/-5% (+/-SD). There was a quantitative recovery of pure RBP added in varying amounts to normal plasma. The immunoassay accurately measured RBP in amounts of 10-100 ng per assay tube. There was no significant difference in the immunoreactivity of apo-RBP as compared to holo-RBP. The mean plasma values (+/-SEM) for a group of 76 normal subjects were 47.2 +/-1.6 mug/ml for males and 41.6 +/-1.6 mug/ml for females. Plasma RBP levels were markedly depressed (15 +/-2.3 mug/ml) in 14 patients with acute viral hepatitis. There was a highly significant correlation between the plasma levels of RBP and of vitamin A in both normal subjects and patients with hepatitis. In all subjects plasma RBP was generally saturated with retinol. The data suggest that under normal circumstances RBP circulates almost exclusively as the holoprotein.