Plasma values of vitamin A and its binding proteins (retinol binding proteins and prealbumin), carotene, total cholesterol and HDL-cholesterol in subjects with breast carcinoma

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 33 subjects with breast cancer and compared to those of a group of healthy subjects previously described. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Mean plasma values of vitamin A, carotene and HDL-cholesterol were lower (P less than 0.01) than in the control group, the same applies to the RBP and PA mean levels (P less than 0.05). On the contrary, the mean value of total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related to RBP and PA. No significant statistical correlation was found between clinical stage and vitamin A plasma levels.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in patients with carcinoma of the cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol of 40 subjects with carcinoma of cervix uteri were determined and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Plasma mean values of carotene, vitamin A, RBP and HDL-cholesterol were lower (P less than 0.01) than in the control group, and the same applies to the PA mean plasma level (P less than 0.05). On the contrary, total cholesterol mean value of the patients resulted to be higher (P less than 0.01) than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the clinical stage and the histological grading and vitamin A plasma levels.     

Quantitation of serum retinol-binding protein by an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent technique for human serum retinol-binding protein (RBP) was developed. The assay detects RBP via a double-antibody (rabbit anti-human RBP) sandwich technique. The antibody is immobilized by passive adsorption to a polystyrene tube; the assay is then carried out by successive additions containing known and unknown amounts of RBP (antigen), alkaline phosphatase linked to the same antibody, and p-nitrophenyl phosphate (substrate). Colorimetric analysis of the hydrolysis of the substrate by the enzyme (indirectly) attached to the antigen is used for RBP quantitation. The intra- and interassay coefficients of variation ranged between 4 and 7 and 9 and 12%, respectively. The assay can be performed in less than 7 h and has a sensitivity in the nanogram range (3–48 ng/ml). RBP content was analyzed in serum and urine samples of 20 healthy donors and 17 patients with renal failure and in 20 serum specimens of patients with liver cirrhosis. Renal patients had higher serum (mean 150, range 50–398 μg/ml) and urine RBP levels (mean 14, range 1–80 μg/ml) than normal donors (mean serum 43, range 30–60 μg/ml; mean urine RBP 0.06, range 0.04 – 0.13 μg/ml). Liver disease patients had lower than normal serum RBP values (mean 22, range 10–43 μg/ml).     

Retinol binding protein 4 levels relate to the presence and severity of coronary artery disease

BackgroundThe previous studies have showed that serum retinol binding protein 4 (RBP4) levels increase in metabolic disorders which are closely associated with cardiovascular diseases (CVD). However, the human studies investigating the role of RBP4 in CVD are conflicted. Therefore, we aimed to evaluate the relationship between RBP4 with the presence and severity of coronary artery disease (CAD) in this study.Methods55 patients with presenting acute coronary syndrome (ACS) and 43 control subjects who had various cardiovascular risk factors with normal coronary artery on coronary angiography were included in this study. The serum RBP4 concentrations were measured using ELISA method, clinically and anatomically score models were used to assess the severity of coronary lesion.ResultsSerum RBP4 levels were significantly higher in patients with ACS compared to the without ACS (68.40 ± 47.94 mg/L vs. 49.46 ± 13.64 mg/L; p = 0.014). RBP4 was correlated with GENSINI and SYNTAX I score (r = 0.286 p = 0.034; r = 0.403 p = 0.002 respectively). However, there was no relationship between RBP4 and GRACE score.ConclusionsThe serum RBP4 levels increase in patients with CAD and its increased levels may be correlated with CAD severity.     

血清视黄醇结合蛋白4与慢性乙肝的相关性分析

目的：观察慢性乙肝患者血清视黄醇结合蛋白4（RBP4）、前白蛋白（PA）、血氨水平,分析RBP4与慢性乙肝的相关性。方法：采集90例慢性乙肝患者分为轻度组26例,中度组34例,重度组30例,另选择30例健康对照组。采用酶联免疫吸附法（ELISA）测定血清RBP4水平,采用免疫透射比浊法测定血清PA水平,用全自动生化分析仪测定血氨水平。结果：血清RBP4、PA在病例组中低于对照组,差异有统计学意义（P〈0.05）,血氨水平在病例组中高于对照组,差异有统计学意义（P〈0.05）。血清RBP4、PA、血氨水平在轻度、中度、重度组内比较P〈0.05,有统计学意义。经Pearson＇s相关分析,血清RBP4与PA浓度呈显著正相关相关（r=0.896,P〈0.01）,与血氨浓度呈显著负相关（r=-0.781,P〈0.01）。结论：RBP4与慢性乙肝存在一定的相关性,与肝脏的损伤程度有关,可作为预测肝脏损伤的血清标志物。     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in healthy adults of the female sex

Plasma vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were examined in healthy adult females. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Vitamin A and carotene mean values resulted as 43.0 +/- 8.2 micrograms/100 ml and 231.9 +/- 69.0 micrograms/100 ml, respectively. RBP and PA values averaged as 4.2 +/- 1.1 mg/100 ml and 29.4 +/- 6.1 mg/100 ml, respectively; whereas HDL-and total cholesterol were 179 +/- 16 mg/100 ml and 57 +/- 8 mg/100 ml. Vitamin A plasma levels were shown to be significantly related (P less than 0.01) to RBP and PA, but not to the other parameters examined (carotene, HDL-and total cholesterol).     

Effect of renal and liver failure on blood levels of vitamin A, its precursor (beta-carotene) and its carrier proteins (prealbumin and retinol binding protein)

Plasma beta-carotene and retinol assay was performed by high pressure liquid chromatography (HPLC) in subjects with chronic renal failure or liver cirrhosis. In the same subjects blood prealbumin (PA) and retinol binding protein (RBP) were determined by immunological technique. A considerable increase of retinol and in a lesser extent of beta-carotene was noted in the blood of patients with renal insufficiency. In cirrhotic patients it was shown a marked decrease both of beta-carotene and retinol plasma concentrations. PA and RBP there were greatly increased in renal failure and decreased in liver cirrhosis. This results suggest that kidney and liver chronic failure interfere with vitamin A metabolism throughout their action on metabolic processes of synthesis and elimination of PA and RBP.     

Behavior of vitamin A, beta-carotene, retinol binding protein and prealbumin in the plasma of hypo- and hyperthyroid subjects

Plasma concentrations of beta-carotene and retinol, determined by HPLC, and of transport proteins, ascertained by immunodiffusion technique, in hypo and hyperthyroid subjects are reported. In hypothyroid subject a considerable increase in carotene was noted. This was not the case for retinol. In hyperthyroids both beta-carotene and retinol levels were found to be normal. Transport protein (PA and RBP) levels were found to be lower only in cases of hyperthyroidism but unchanged for hypothyroids. According to the Authors the results show that the alteration in plasma carotene levels to be found in hypothyroid subjects is not the direct consequence of a lack of thyroid hormone in the metabolism of vitamin A but the indirect effect of thyroid disease.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.     

Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.

The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.     

Demonstration of immunoreactive retinol-binding protein and prealbumin in developing chicken embryo.

The immunoreactive retinol-binding protein (RBP) and prealbumin (PA) were identified in chicken embryo by the method of double immunodiffusion using antisera against purified chicken serum RBP and PA, respectively. The embryonic RBP studied by a fluorospectrophotometric analysis showed presence of vitamin A (retinol) within the molecule. The RBP and PA fractionated on a column of Sephadex G-200 had molecular weight of approximately 20,000 and 56,000, respectively. RBP and PA formed a complex with vitamin A which had a molecular weight of approximately 76,000. The developmental changes of RBP and PA in the chicken embryo were determined in the eye, brain, serum and liver by the single radial immunodiffusion. In the brain and eye, the maxima for the concentration of RBP and PA were detected at day 6 for RBP, and day 6 and day 13 for PA during development. However, these proteins were not detected in the tissues of young chicken. The concentration of the serum embryonic RBP and PA showed a maximum at day 6. With regard to the liver, the PA was observed in the embryo only at day 13, but the RBP only after hatching.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and cholesterol-HDL in patients with dysplasia of the uterine cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 19 female subjects with cervical dysplasia and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion and HDL- and total cholesterol by enzymatic colorimetry. Plasma mean values of vitamin A and HDL-cholesterol were lower (P less than 0.05 and P less than 0.01, respectively) than in the control group. On the contrary total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the severity of the dysplasia and vitamin A plasma levels.     

Assay of ethynyloestradiol in human serum and its binding to plasma proteins

A radioimmunoassay for estimating both unconjugated and conjugated ethinyl estradiol (EE) in serum is described, along with a report of levels of EE attained after its administration and the binding of EE in plasma, as determined by this radioimmunoassay. Mean values for concentration of free EE were 38-87 pg/ml, 1-4 hours after administration, and by 24 hours levels in most subjects were below the sensitivity level of this assay, which is less than 25 pg/ml. Conjugated EE concentration in serum was considerably higher, with mean values of 370-770 pg/ml 1-4 hours after ingestion, decreasing slowly to mean values of 285 pg/ml at 8 hours and 100 pg/ml at 24 hours postadministration. Total EE (conjugated plus unconjugated) had a mean half-life of 3.8 hours during the period 2-8 hours after administration and 10.7 hours for the period 8-24 hours postadministration. EE in plasma was shown, by equilibrium dialysis, gel filtration on Sephadex G200, and polyacrylamide gel electrophoresis, to bind only to albumin, and no specific binding of EE occurred. The apparent association constant for the binding of EE to human serum albumin was 1.7 times 10 5 liter per mol.     

Polyamine concentrations in red cells and urine of patients with chronic renal failure

Spermidine and spermine concentrations were measured in 6 healthy subjects, 18 patients with chronic renal failure and 6 patients undergoing maintenance hemodialysis. In nondialyzed patients with advanced renal failure (serum creatinine levels greater than 6 mg %), red cell spermidine concentrations were significantly higher than in normal subjects (54.8±14.5 vs. 24.8±63 SD nmoles/ml packed cells). However red cell spermine concentrations were unchanged as compared to normal subjects (18.7±7.3 vs. 12.4±3.4 nmoles/ml packed cells). In patients with serum creatinine levels below 6 mg%, neither red cell spermidine or spermine concentrations were significantly different from normal subjects. There was a significant correlation between red cell spermidine values and both serum urea and serum creatinine levels, but no correlations were observed for red cell spermine. Red cell spermidine values were also significantly higher in patients undergoing maintenance hemodialysis than in normal subjects. In each patient, red cell spermidine concentrations were no different after a hemodialysis treatment than immediately prior to dialysis. In urine, excretion rates of polyamines as well as the precursor diamine, putrescine, were lower in patients with chronic renal failure than in normal subjects. Hence in renal failure, one factor contributing to the accumulation of spermidine in red cells would appear to be a decrease in the urinary excretion of polyamines.     

Haematological values in pregnant women in Port Harcourt, Nigeria II: Serum iron and transferrin, total and unsaturated iron binding capacity and some red cell and platelet indices

Previous studies on the normal values of serum iron, unsaturated iron binding capacity, total iron binding capacity, serum transferrin, percent transferrin saturation, red cell distribution width, and various platelet indices: Platelet count, mean platelet volume, platelet distribution width, plateletcrit and platelet larger cell ratio in pregnant subjects in Nigeria are relatively scanty. Present study aims to determine the values of these parameters in apparently healthy pregnant subjects residing in Port Harcourt south eastern Nigeria; and help establish normal reference ranges of these parameters for the population under reference. Cross sectional prospective study involving 220 female subjects attending for the first time, the ante-natal clinics of a tertiary health care facility in Port Harcourt. Subjects were divided into 73, 75 and 72 subjects in the first, second and third trimester of pregnancy respectively. Serum iron and unsaturated iron binding capacity, red cell distribution width, platelet count and platelet distribution width were determined by automated methods; total iron binding capacity, serum transferrin concentrations, percent transferrin saturation, mean platelet volume and plateletcrit were calculated using appropriate formulas. The values of serum iron, unsaturated iron binding capacity, total iron binding capacity and serum transferrin concentrations were found to show significant variations between the various trimesters of pregnancy. However, while serum iron showed significant decreases during pregnancy; unsaturated iron binding capacity, total iron binding capacity and serum transferrin concentrations were found to show significant increases during pregnancy amongst our subjects. By contrast the values of red cell distribution width, platelet count, mean platelet volume, platelet distribution width, plateletcrit and platelet larger cell ratio did not show any significant differences at the different trimesters of pregnancy in our subjects. The present study reports, for the first time, normative values for these parameters in apparently healthy pregnant subjects in Port Harcourt south eastern Nigeria. Apparently, increases in unsaturated and total iron binding capacity and serum transferrin values seen amongst our subjects with increasing gestation may perhaps be a mechanism to ensure a fetal adequate iron delivery on account of the decreasing serum iron concentration with gestation in our subjects. The study suggests that values of serum transferrin are perhaps a more useful screening tool for iron deficiency anemia during pregnancy amongst our subjects.     

Vaginal hydrolysis of retinyl acetate: increase in plasma retinol and retinol binding protein in women with cervical dysplasias

We previously reported the clinical feasibility of a Phase I trial involving the topical administration of a RA gel applied cervicovaginally in women with mild or moderate cervical dysplasia. Now, we report hydrolysis and systemic absorption of the RA gel from the vagina. HPLC analysis of samples of residual gel obtained from the cervical canal after topical bolus application indicate that the RA undergoes prompt in vivo hydrolysis yielding retinol as a major metabolite. Venous blood samples of 41 subjects, who self-administered a RA gel, were analyzed for plasma retinol and RBP concentrations prior to and upon completion of a 7-day treatment course and upon return for follow-up examinations. An increase in both the concentrations of plasma retinol and RBP were detected after topical application of the RA gel. These elevated values receded after the gel administration was discontinued. No significant changes were observed in plasma retinol or RBP concentrations in placebo-treated subjects. The efficacy of RA as a chemopreventive agent in treating cervical dysplasias remains to be determined.     

Interactions of retinol with binding proteins: implications for the mechanism of uptake by cells

The kinetic parameters of the interaction of retinol with retinol binding protein (RBP) were studied. The rate constant for association of retinol with the protein (ka) was found to be 1.5 X 10(6) M-1 min-1. The rate constant for dissociation (kd) from the protein was determined by studying the transfer of retinol from RBP to lipid bilayers. It was found that such transfer proceeds via the aqueous phase and its rate-limiting step is the dissociation of retinol from the binding protein. The rate of transfer therefore represents the rate of dissociation. The kd was 0.112 min-1. These values were validated further by the following consideration. The equilibrium dissociation constant of RBP and retinol can be calculated from the expression Kd = kd/ka. The calculated value was 7.5 X 10(-8) M. Kd was also measured directly by fluorometric titration and was found to be 7 X 10(-8) M. The relative avidities of retinol for RBP, the complex RBP-transthyretin (RBP-TTR), and serum albumin were also studied. It was found that binding of RBP to TTR increased its avidity for retinol by about 2-fold. The avidity of albumin for retinol was 30-fold lower than that of RBP. The data imply that retinol spontaneously and rapidly dissociates from sites on binding proteins, which indicates that the vitamin can freely move in vivo between physiologic compartments with avidities for it.     

Retinol to retinol-binding protein (RBP) is low in obese adults due to elevated apo-RBP

Elevated serum retinol-binding protein (RBP) concentration has been associated with obesity and insulin resistance, but accompanying retinol values have not been reported. Assessment of retinol is required to discriminate between apo-RBP, which may act as an adipokine, and holo-RBP, which transports vitamin A. The relations between serum RBP, retinol, retinyl esters, BMI, and measures of insulin resistance were determined in obese adults. Fasting blood (> or =8 h) was collected from obese men and women (n = 76) and blood chemistries were obtained. Retinol and retinyl esters were quantified by HPLC and RBP by ELISA. RBP and retinol were determined in age and sex-matched, nonobese individuals (n = 41) for comparison. Serum apo-RBP was two-fold higher in obese (0.90 +/- 0.62 microM) than nonobese subjects (0.44 +/- 0.56 microM) (P < 0.001). The retinol to RBP ratio (retinol:RBP) was significantly lower in obese (0.73 +/- 0.13) than nonobese subjects (0.90 +/- 0.22) (P < 0.001) and RBP was strongly associated with retinol in both groups (r = 0.71 and 0.90, respectively, P < 0.0001). In obese subjects, RBP was associated with insulin (r = 0.26, P < 0.05), homeostatic model assessment of insulin resistance (r = 0.29, P < 0.05), and quantitative insulin sensitivity check index (r = -0.27, P < 0.05). RBP was associated with BMI only when obese and nonobese subjects were combined (r = 0.25, P < 0.01). Elevated serum RBP, derived in part from apo-RBP, was more strongly associated with retinol than with BMI or measures of insulin resistance in obese adults. Investigations into the role of RBP in obesity and insulin resistance should include retinol to facilitate the measurement of apo-RBP and retinol:RBP. When evaluating the therapeutic potential of lowering serum RBP, consideration of the consequences of vitamin A metabolism is paramount.     

A slab gel electrophoresis technique for measurement of plasma retinol-binding protein, cellular retinol-binding and retinoic-acid-binding proteins in human skin

At least four different proteins that bind retinoids could be present in a vitamin A target tissue like the skin. In order to separate cellular retinoid-binding proteins (CRBP and CRABP) from serum retinol-binding protein (RBP) and albumin, a one-step procedure was devised. The technique is based on slab polyacrylamide gel electrophoresis (PAGE) of the extracted proteins incubated with tritiated retinoids. The procedure was used to study binding proteins in the skin. The results show that epidermal extracts (the epithelial part of the skin) contain no RBP activities whereas dermal extracts (the mesenchymal part of the skin) contain 1.6 +/- 0.81 pmol/mg protein of RBP. This technique further showed higher levels of CRABP in both epidermal (9.05 +/- 1.16 pmol/mg protein) and dermal (1.5 +/- 0.54 pmol/mg protein) extracts than those previously determined by other less specific techniques. On the other hand CRBP levels were found to be lower in the two tissues (epidermis 0.2 +/- 0.1 pmol/mg and dermis 0.12 +/- 0.05 pmol/mg protein). New conditions to measure specifically CRABP with the charcoal/dextran technique could be developed and analyzed by the PAGE technique; a dissociation constant of 13.7 nM was then calculated for epidermal CRABP. This PAGE technique appears to be the most appropriate method for the study of retinoid-binding proteins including RBP in human skin.     

Subcellular localization in normal and vitamin A-deficient rat liver of vitamin A serum transport proteins,albumin, ceruloplasmin and class I major histocompatibility antigens

The subcellular localization in rat liver cells of retinol-binding protein (RBP), prealbumin, ceruloplasmin, albumin, and class I transplantation antigen chains was investigated by radioimmunoassay determinations. The concentration of RBP was high in the rough and smooth endoplasmic reticulum (SER). The relative concentrations of prealbumin, ceruloplasmin and albumin were similar in the endoplasmic reticulum fractions and in the Golgi fraction. Neither of the proteins were found in significant amounts in the post-microsomal supernatant nor in the plasma membrane. The concentrations of the class I transplantation antigen chains were higher in the Golgi fraction than in the endoplasmic reticulum fractions. In the rough endoplasmic reticulum (RER) fraction ceruloplasmin and the class I antigens partially interact with high-molecular weight (MW) components, presumably membrane-bound glycosyltransferases. RBP, prealbumin and albumin seemed to be present in free form within the microsomal lumen. In vitamin A deficiency the RBP and to a lesser extent the prealbumin concentrations in the endoplasmic reticulum fractions were significantly increased, as compared to fractions from normal livers. This suggests that the presence of vitamin A is a prerequisite for the transport of RBP from the endoplasmic reticulum to the Golgi complex. The intracellular concentrations of albumin and ceruloplasmin were not significantly altered by vitamin A deficiency. In contrast, the amounts of the class I antigen heavy chains were found to be increased.