The Determination of Erythrocyte Folate Concentration Using a Two-Phase Ligand-binding Radioassay

The concentration of folate in erythrocyteswas determined using a two-phase ligand-binding radioassay procedure described previously for measuring serum folate. Themean (± SD) folate concentration in erythrocytes of 20 normal subjects was 210 ± 57ng/ml. In 12 patients clinically folatedeficient who had normal serum B₁₂concentration, the mean (± SD) erythrocytefolate was 71 ± 39 ng/ml. Incubation of thelysed erythrocytes for 2 hr prior to boilingincreased the radioassayable folate. Theradioassayable folate decreased rapidly ifthe whole blood was stored at 4°C withoutascorbate. Extracts of blood prepared withascorbate could be stored at -20°C forseveral days. The radioassayable concentration of erythrocyte folate was similarto the values obtained using Lactobacilluscasei when the concentration was 200 ng/ml or less. With values higher by L. casei,the radioassayable folate was significantlylower even though the normal and folate-deficient groups were distinctly separated.This radioassay provides a rapid and reliablemethod of measuring erythrocyte folate, aparameter which reflects folate stores morereliably than serum folate concentration.

Submitted on July 27, 1973 Accepted on August 20, 1973     

Recent trends in use of herbal and other natural products

BACKGROUND: The benefits of herbal and other natural products (dietary supplements) are increasingly cited in the media. Dramatic increases in use reported during the last decade have led to growing concerns about efficacy and safety. METHODS: To determine which dietary supplements American adults use, whether the prevalence has continued to increase in recent years, and whether popularity of individual supplements has changed, demographic information and details of use of all medicines and dietary supplements in the preceding week were obtained by telephone interview from February 1998 through December 2002 from households in 48 contiguous states and the District of Columbia. Participants included randomly selected residents of households with telephones; compared with 2000 US Census data, participants were representative of the US population. The main outcome measure was the weekly prevalence of dietary supplement use, alone or in a multicomponent product. RESULTS: There were 8470 subjects 18 years or older. The annual prevalence of dietary supplement use increased from 14.2% in 1998-1999 to 18.8% in 2002. Although use did not change among younger subjects, it doubled for men and women 65 years or older. Use of Ginkgo biloba and Panax ginseng declined during the study, while lutein use increased dramatically, because of its addition to multivitamin products. The overall 2002 prevalence excluding lutein use was 13.9%. CONCLUSIONS: The popularity of specific supplements has varied over time and differs according to age and sex. The sharp increase in supplement use in the 1990s appears to have slowed. However, the addition of supplements, such as lutein and lycopene, to mainstream multivitamins has become an important source of exposure.     

Prevalence of oral lesions and percent CD4+ T-lymphocytes in HIV-infected children on antiretroviral therapy

This study examined prevalence of oral lesions and how it relates to CD4 percentages in vertically infected children with HIV undergoing combination antiretroviral therapy. One hundred two HIV-infected children between the ages of 3 and 15 years attending a specialized pediatric outpatient clinic were examined for oral lesions, and their CD4 percent and viral load extracted from their medical records. Of the 102 HIV-infected children, 69% had evidence of oral pathology and 31% were disease free. The proportion with disease was: 20.6% had conventional gingivitis, 19.6% had dental caries in their primary and permanent teeth combined, 13.7% had depapillated tongue, 3.9% had early childhood caries, 2.9% had oral candidiasis, 2% had bilateral enlarged parotid gland, 1% had median rhomboid glossitis, 1% had enlarged cervical lymph nodes and 2% had other developmental abnormalities. In the group with no evidence of suppression 15% had gingival lesion, 14% tongue lesion, and 1% parotid enlargement, and in the severe suppression group 55% had gingival lesion, 45% had tongue lesion, 9% had enlarged cervical lymph nodes, and another 9% had parotid gland enlargement. The association between conventional gingivitis and low CD4 percent was statistically significant (p = 0.001). Compared to previous studies, overall prevalence estimates of oral lesions in this study was low. Children with low CD4 percent had more oral lesions, consistent with results from other HIV studies.     

Unrelated donor marrow transplantation in children

Eighty-eight children 0.5 to 17 years of age (median, 9 years of age) received an unrelated donor marrow transplant for treatment of chronic myeloid leukemia (CML; n = 16), acute lymphoblastic leukemia (ALL) in first or second remission (n = 15) or more advanced stage (n = 28), acute myeloid leukemia (AML; n = 13), or other hematologic diseases (n = 16) between June 1985 and April 1993. All patients were conditioned with cyclophosphamide and total body irradiation and received a combination of methotrexate and cyclosporine as graft-versus-host disease (GVHD) prophylaxis. Fourty-six patients received transplants from HLA-identical donors and 42 patients received transplants from donors who were minor-mismatched at one HLA-A or B or D/DRB1 locus. The Kaplan-Meier estimates of disease-free survival and relapse were 75% and 0% for patients with CML, 47% and 20% for ALL in first or second remission, 10% and 60% for ALL in relapse or third remission, 46% and 46% for AML in first remission (n = 1) or more advanced disease (n = 12), and 29% and 69% for other diseases. HLA disparity was not significantly associated with lower disease-free survival, but the results suggest more relapses in HLA-matched recipients and there was significantly more transplant-related mortality in mismatched recipients (51% v 24%, P = .04). Most deaths were due to infections associated with acuteor chronic GVHD and occurred within the first 2 years after transplantation. Granulocyte engraftment occurred in all evaluable patients. Sixty-three percent of HLA-matched and 57% of HLA- mismatched recipients were discharged home disease-free at a median of 98 and 103 days, respectively, after transplantation (P = not significant [NS]). The incidence of grades II-IV acute GVHD was 83% in HLA-matched and 98% in HLA-mismatched recipients (P = .009). The incidence of chronic GVHD was 60% in HLA-matched and 69% in HLA- mismatched recipients (P = NS). One or multiple late adverse events such as cataracts, osteonecrosis of the hip or knee, restrictive or obstructive pulmonary disease, and hypothyroidism have occurred in 11 of 33 (33%) surviving patients. Immunosuppression was discontinued in 58% of surviving patients, including all 12 patients surviving more than 3.2 years, all of whom have a Lansky or Karnofsky score of 100%.(ABSTRACT TRUNCATED AT 400 WORDS)     

A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/microg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status.     

Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.     

Acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas and other malignancies in patients with hemophilia

Non-Hodgkin's lymphoma (NHL) is the most common human immunodeficiency virus (HIV)-associated malignancy in hemophiliacs. We studied the incidence and clinicopathologic features of NHL in 3,041 hemophiliacs followed at 18 US Hemophilia Centers between 1978 and 1989. Of the 1,295 (56.6%) who were HIV(+), 253 (19.5%) developed acquired immunodeficiency syndrome (AIDS), of whom 14 (5.5%) developed NHL. Three NHL occurred in HIV(-) hemophiliacs, for a 36.5-fold greater risk in HIV(+) than HIV(-) hemophiliacs (P < .001). The NHL incidence rate was 29-fold greater than in the US population by Surveillance, Epidemiology, and End Results (SEER) estimates (P < .001). Between 0 and 4 lymphomas have been observed per year between 1978 and 1989. At presentation 13 (92.9%) of the HIV(+) NHL were extranodal. Ten were stage IV, 1 stage II, and 3 stage IE. Ten (71.4%) were high-grade, 3 (21.4%) intermediate-grade, and 1 (7.1%) was a low-grade B-cell lymphoma. Epstein-Barr virus (EBV) DNA was detected in 36% by in situ hybridization, including one central nervous system (CNS) lymphoma. The mean CD4 cell count at NHL diagnosis was 64/mm3, the mean latency from initial HIV infection was estimated to be 59 months, and the median survival was 7 months. The incidence of basal cell carcinoma in HIV(+) hemophiliacs was 18.3-fold greater than in HIV(-) hemophiliacs (P < .001) and 11.4-fold greater than in the US population (P < .001). In conclusion, incidence rates of NHL and basal cell carcinoma in HIV(+) hemophiliacs are significantly increased over rates in HIV(-) hemophiliacs and over rates in the US population. Clinicopathologic presentation of NHL in HIV(+) hemophiliacs is similar to that in HIV(+) homosexual men.     

Evaluation of platelet response to different clopidogrel dosing regimens in patients with acute coronary syndrome in clinical practice

High-post clopidogrel platelet reactivity in acute coronary syndrome (ACS) patients is associated with adverse outcomes and may be related to clopidogrel dosing. Clinical studies evaluating different clopidogrel doses have resulted in conflicting conclusions. Clopidogrel dosing regimens have evolved over time, enabling us to evaluate platelet reactivity in real-life ACS patients undergoing percutaneous coronary intervention and treated with three different clopidogrel doses. Platelet reactivity was assessed with light transmitted aggregometry on the third day post clopidogrel loading in 404 consecutive ACS patients. Of them, 198 were treated with a standard regimen (300?mg loading, 75?mg/day maintenance dose), 95 with a high loading regimen (600?mg loading, 75?mg/day maintenance dose) and 111 with a high loading/high maintenance regimen (600?mg loading, 150?mg/day maintenance). Compared with the standard regimen, the high loading regimen resulted in significantly lower mean platelet reactivity to adenosine diphosphate (ADP) with a lower proportion of patients exhibiting clopidogrel non-responsiveness (11% vs. 28%, p?=?0.004). Compared with the high loading regimen, the high loading/high maintenance regimen resulted in significantly lower mean platelet reactivity to ADP, but without a further drop in the number of non-responders (8.1% vs. 11%, p?=?0.16). In conclusion, greater overall inhibition can be achieved with higher loading and maintenance doses in ACS patients. However, despite high clopidogrel doses, a sizable proportion of patients remained “resistant” to the effects of clopidogrel.