Plasma homocysteine and folate levels and dietary folate intake in adolescents and young adults who underwent kidney transplantation during childhood.

BACKGROUND: Hyperhomocysteinemia (hyper-Hcy) is an important and reversible cardiovascular disease risk factor. We examined the prevalence of hyper-Hcy, plasma folate levels, and dietary folate intake in adolescents and young adults who had undergone kidney transplantation during childhood to assess the necessity for managing dietary folate. METHODS: This cross-sectional study was performed in 89 kidney transplant recipients (age at kidney transplantation: 12.6 ± 4.1 years; age during study: 21.2 ± 5.5 years). Hyper-Hcy and plasma folate deficiency were defined as plasma homocysteine (Hcy) >15 nmol/ml and plasma folate <3.0 ng/ml, respectively. RESULTS: Of the patients, 60 (67.4 %) had hyper-Hcy and 14 (15.7 %) had plasma folate deficiency. Plasma homocysteine levels correlated negatively with estimated glomerular filtration rate (eGFR; r = -0.565, p < 0.01) and plasma folate levels (r = -0.434, p < 0.01). For determinants of plasma homocysteine levels, a priori selected variables included kind of calcineurin inhibitor, age at kidney transplantation, pretransplant duration of dialysis, time since transplantation, age at examination, eGFR, and plasma folate. Stepwise multiple linear regression analysis revealed eGFR and plasma folate levels as significant independent variables influencing plasma homocysteine levels. Dietary folate intake in 11 of 16 patients (66.8 %) with eGFR ≥ 60 ml/min/1.73 m(2) was below the recommended dietary allowance for Japanese. CONCLUSIONS: The prevalence of hyper-Hcy and plasma folate deficiency, as well as the low dietary folate intake, suggest that dietary management of folate is necessary for adolescents and young adults who have undergone kidney transplantation during childhood.

[Nutrient intake and dietary habit in pediatric, adolescent, and young adult kidney transplant recipients].

AIM: Dietary advice by dietitians based on various factors of nutrient intake and dietary habit is important for preventing postoperative complications in adult kidney transplant recipients, but little is known about whether such advice is also important for pediatric, adolescent, and young adult kidney transplant recipients. The purpose of this study was to examine the nutrient intake profiles and dietary habits of these recipients and to ascertain the need for dietary advice by dietitians. SUBJECTS AND METHODS: This study involved 22 kidney transplant recipients with an estimated glomerular filtration rate> or =60 mL/min/1.73 m2 with no dietary restrictions. Nutrient intake was measured using a food frequency questionnaire based on food groups and evaluated using the reference values given in the Dietary Reference Intakes for Japanese (DRIs-J)for 2010 and the daily average intake of the National Health and Nutrition Survey Japan, 2010. RESULTS: The mean age of the patients at the time of enrollment was 17.6+/-4.4 years(8.7 28.5 years). Energy and carbohydrate intakes were below the DRIs-J reference value in 18 patients(81.8 %), and the ratio of total fat to total energy was above the DRIs-J reference value in 20 patients(90.9 %). Vitamin B, vitamin B6, magnesium, and zinc intakes were below the DRIs-J reference value in all patients (100 %). Food group intakes that were less than 75 % of the values reported in the National Health and Nutrition Survey Japan were pulses and algae in 18 patients(81.8 %), green and yellow vegetables in 14 patients(63.6 %), other vegetables in 19 patients(86.4 %), and nuts and seeds in 16 patients(72.7 %). CONCLUSION: This study suggests that dietary advice by dietitians is necessary for pediatric, adolescent, and young adult kidney transplant recipients to improve their nutrient intake and dietary habits.

Function of mutant (G1144A) tissue-nonspecific ALP gene from hypophosphatasia.

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In our previous study, we found the novel point mutations (G1144A and T979C) from the genomic TNSALP gene of a patient with childhood HOPS. In this study, we have characterized the protein translated from the mutant G1144A gene. Wild-type and G1144A mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h or 72-h transfection, cells were collected and homogenized using polytron homogenizer. After centrifugation at 10,000 g for 10 minutes, the supernatant was assayed. ALP activity was determined with 10 mM of p-nitrophenylphosphate as a substrate in 100 mM of 2-amino-2-methyl-1,3-propanediol-HCl buffer containing 5 mM of MgCl2. ALP activity of cells transfected with the mutant cDNA (G1144A) plasmid after 48-h or 72-h transfection exhibited 0.063 +/- 0.012 U/mg and 0.054 +/- 0.012 U/mg, respectively. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 2.7% and 1.7%, respectively. These values were not significantly different from those found with mock-transfected cells, that is, 2.5% and 1.5%, respectively. This study indicated that the mutation (G1144A) produced the inactive ALP enzyme and would be a disease-causing mutation of the childhood-type HOPS.

The mutant (F310L and V365I) tissue-nonspecific alkaline phosphatase gene from hypophosphatasia.

Hypophosphatasia (HOPS) is a heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity and premature loss of deciduous teeth. In a previous study, we detected missense mutations in the TNSALP gene of a patient who inherited the F310L and the V365I mutation with severe periodontitis and childhood HOPS. Expression of the mutant V365I TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant had undetectable ALP activity. In the present study, we characterized another ALP enzyme translated from the mutant F310L and compared it with the ALP in the patient's serum. The COS-1 cells transfected with the F310L and co-transfected with F310L and V365I (F310L/V365I) exhibited levels of 67% and 31%, respectively, with the enzymatic activity of the wild-type taken as 100%. In the thermostability test, TNSALPs in the COS-1 cells transfected with the mutant F310L or F310L/V365I were significantly more heat labile compared with that of the wild-type. Moreover, ALP from the patient's serum was also more heat labile than normal ALP. These results suggest that the protein translated from the mutant F310L, in addition to the mutant V365I, may be responsible for the expression of symptoms of the childhood-type HOPS.

Characterization of the mutant (A115V) tissue-nonspecific alkaline phosphatase gene from adult-type hypophosphatasia.

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. To date, various mutations in the TNSALP gene have been identified. Especially, A115V located in exon 5 has been detected in a Japanese patient with severe periodontitis and adult-type HOPS. In this study, we have characterized the protein translated from the mutant A115V gene. Wild-type and A115V mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h transfection, the cells were subjected to assay ALP activity. In order to identify possible dominant effect of the mutation, we performed co-transfections of wild-type and mutated cDNA, and evaluated the residual activities of each mutation. Detection of TNSALP synthesized by COS-1 cells transfected with the wild- or the mutated-type was also performed by using an immunofluorescent method. ALP activity of cell transfected with the mutant cDNA (A115V) plasmid after 48-h transfection exhibited 0.399+/-0.021 U/mg. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 16.9%. When co-transfected this mutant showed no inhibition of the wild-type enzyme. TNSALP in COS-1 cells with transfected with the mutant exhibited strong fluorescence at the surface of cells as wild-type. This study indicated that the mutant (A115V) TNSALP gene produced the defective ALP enzyme and it could be recessively transmitted and be a disease-causing mutation of the adult-type hypophosphatasia.

Phosphate depletion enhances bone morphogenetic protein-4 gene expression in a cultured mouse marrow stromal cell line ST2.

Alkaline phosphatases (ALPs) are glycosylated, membrane-bound enzymes that hydrolyze various monophosphate esters at an optimum high pH and are present in nearly all living organisms. In Escherichia coli, extracellular phosphate (Pi) limitation induces the ALP gene, indicating a role of extracellular Pi in ALP gene regulation. However, little is known about similar mechanisms in mammalian cells. Previously, we reported that Pi starvation increased the tissue-nonspecific ALP (TNSALP) activity and regulated its expression in the mouse stromal cell line ST2, derived from mouse bone marrow. In the present study, we further examined the effects of Pi starvation on the mechanism of TNSALP induction. The specific activity of TNSALP increased markedly after treatment by Pi starvation for 5 days and RT-PCR analysis revealed that the mRNA of the bone morphogenetic protein-4 (BMP-4) gene was highly stimulated. The combination of Pi depletion and mouse BMP-4 receptor IA/Fc chimera down-regulated the TNSALP activity. These results indicated that Pi depletion stimulates the TNSALP activity for the Pi supplementation, and that this system may involve the signaling pathway of the BMP-4 gene at the transcription level.