ALBERT

Description: BACKGROUND: We have recently identified a SNP (rs5988) in the gene encoding the factor XIII A chain (F13A) that is highly associated with risk for priapism in SCD. The P value for the comparison of G/G vs. C/G was 0.009, and the odds ratio for having priapism was 2.52 (CI 1.27-5.03) for men with the G/G F13A genotype (encoding FXIIIA 651E) compared to those with the C/G genotype (expressing FXIIIA 651Q/E). We hypothesized that this polymorphism leads to abnormal FXIII-mediated crosslinking and increased heterologous blood cell aggregates. The mechanism whereby heterologous blood cell aggregates form has only been partially elucidated to date. Brittain et al. (2008) showed that fibronectin is detectable within aggregates and plays a critical role in RBC-monocyte interactions, but it is unknown how fibronectin is recruited. We propose that aggregates form via interactions between fibrin(ogen), other plasma proteins (e.g., fibronectin, laminin), and blood cells during blood stasis in the penis during erection. These aggregates might then obstruct vessels, preventing blood egress, and result in SCD-associated priapism. METHODS: We quantitated circulating heterocellular aggregates using flow cytometry and directly labeled antibodies against CD45 (pan-leukocyte), CD235a (glycophorin A), CD41a (platelet GPIIb), and CD14 (monocytes and granulocytes). Heterocellular aggregates were defined as events simultaneously expressing CD45 and CD235a in whole anticoagulated blood. Events were analyzed by forward and side scatter as well as immunostaining characteristics (Canto II flow cytometer, Becton Dickinson, San Jose). Genotypes were ascertained by SNP genotyping using predesigned and custom Taqman SNP Genotyping Assays (ThermoFisher Scientific, Foster City, CA). Mean values for different genotypes were compared using two-tailed t-tests; FXIII activity was compared using 2-way ANOVA. RESULTS: As previously described, heterocellular aggregates occurred more frequently in blood samples from SCD subjects than from HBAA controls. Among SCD subjects, the % WBCs found in aggregates ranged from 14.9% to 88.5% (mean 58.85%). Specifically, lymphocytes and monocytes were found significantly more frequently in aggregates in HbSS compared to HbAA samples (p=0.042 and 0.025, respectively). When analyzed by F13A genotype, we also found that aggregates containing monocytes and lymphocytes were significantly more numerous in individuals with the GG (priapism risk) genotype than with the CC (low-risk) genotype (p = 0.008 and 0.015, respectively). We then tested recombinant (r) FXIIIA isoforms corresponding to the two alleles of F13A for their ability to bind to and crosslink both fibrinogen and fibronectin. Comparison of their ability to bind fibrinogen, fibrin and fibronectin showed no significant differences between the two rFXIII isoforms after activation. However, activated FXIIIA 651E (G allele) crosslinked both fibrinogen and fibronectin significantly more quickly than did activated FXIIIA 651Q (C allele) (p = 0.006 and p = 0.012, respectively), thus suggesting that the G allele might be associated with greater amounts of crosslinked fibrin(ogen) and fibronectin in the circulation to promote aggregate formation. CONCLUSIONS: Our study has demonstrated that the F13A G allele at rs5988 carries a higher risk for SCD-related priapism and is associated with increased involvement of lymphocytes and monocytes in heterocellular aggregates. A possible mechanism is suggested by our observation that rFXIIIA protein encoded by the G allele more rapidly crosslinks fibrinogen and fibronectin than that encoded by the C allele. We theorize that the G genotype may be related to greater fibrin and fibronectin crosslinking, thus promoting the formation of circulating heterocellular aggregates. Disclosures No relevant conflicts of interest to declare.

Description: No evidence of vitamin B12 deficiency or of any mechanism which might lead to vitamin B12 deficiency, such as defective absorption or increased urinary or fecal excretion of the vitamin, has been found in iron-deficient subjects in whom gastric acid secretion and gastric biopsies were normal. It is concluded that when vitamin B12 deficiency occurs in iron-deficient subjects it is the result of gastric atrophy. An unexplained finding was delayed disappearance of an intravenous dose of Co58-B12 from the plasma in iron-deficient subjects.

Description: Iron absorption measurements have been made in 131 individuals relating the absorption of nine different foods tagged biosynthetically with radioiron. Relatively low absorption, ranging from 1.7-7.9, was found with wheat, corn, black beans, lettuce and spinach. Higher values of from 15.6-20.3 were observed with soybeans, fish, veal and hemoglobin. When these values were related to the absorption of ferrous ascorbate determined simultaneously in each subject, food iron absorption could be predicted over the spectrum of normal to iron deficient states.

Description: A collaborative study of nutritional anemia in third trimester pregnancy was performed in seven Latin American countries. Laboratory measurements included hemoglobin level, mean corpuscular hemoglobin concentration (MCHC), serum iron and iron-binding capacity, serum folate, vitamin B12 and albumin. Iron deficiency (transferrin saturation below 15%) was found in 48% of pregnant women, as compared with 21% of nonpregnant females and 3% of male controls of comparable age. The prevalence of folate deficiency (serum folate below 3 ng/ml.) was 10%, 10% and 9% in these three groups, respectively. Vitamin B12 deficiency (serum level below 80 pg/ml.) was found in 15% of pregnant women, but in less than 1% of both control groups. Anemia, as defined by current WHO criteria, was found in 38.5% of pregnant women, 17.3% of nonpregnant women and 3.9% of men. Analysis of the frequency distribution for hemoglobin levels, based on a Gaussian distribution in normal subjects, suggested that a large portion of subjects considered anemic by WHO criteria were normal and that the true incidence of anemia in pregnant and nonpregnant females was 22 and 12% respectively. Correlation analysis indicated that iron deficiency was of major importance as a cause of anemia, while folate lack was contributory only in pregnancy; no relationship could be demonstrated between vitamin B12 deficiency and anemia.

Description: Sources of variation in iron absorption measurements and methods of controlling them have been reviewed. There are marked differences in the mean level of absorption in different individuals and appreciable day-to-day variation in absorption in the same individual. Intersubject variations can be reduced by the selection of individuals with a more uniform iron requirement, but large variations remain which can be dealt with only by applying appropriate statistical methods to the skewed data obtained and by studying sufficient subjects to provide statistical validity to the results. Comparative studies of the availability of food iron are best carried out against a reference standard in the same subject, and intrasubject variations can be reduced by multiple dose administration.

Description: The assimilation of fortification iron has been studied in 228 subjects by the extrinsic tag method. The first study was made comparing the absorption from ferric and ferrous iron salts given with a complete meal. Labeled wheat enriched with 2 mg of iron as ferric chloride was given with a complete meal, and the same study was repeated in the same subjects with wheat enriched with ferrous sulfate. The same protocol was followed in the second study, but the meal contained no meat. Ferric chloride was absorbed, as well as ferrous sulfate in both studies. Multiple absorptions were performed from test meals containing foods of either animal or vegetal origin tagged biosynthetically with 55Fe (intrinsic tag) to which was mixed iron as 59FeCl3 (extrinsic tag) in doses from 0.5 to 60 mg of iron. With test meals of labeled maize given along or with unlabeled meat, the ratio of extrinsic to intrinsic tag absorption was close to unity along a wide range of absorption percentage and was independent of the dose of iron fortification administered. It is concluded that fortification iron added as a soluble iron salt mixed with the nonheme iron pool of the diet and its absorption reflects the absorption of the nonheme iron. Similar studies in which increasing doses of 59FeCl3 were added to a test meal of tagged animal meat showed a progressive fall in the ratio of extrinsic to intrinsic tag absorption, indicating that assimilation of dietary iron occurs from two independent pools—heme and nonheme iron. Absolute absorption of fortificaiton iron added to maize increased from 0.07 to 0.34 mg iron with an increase in the dose of nonheme iron from 2.5 to 62 mg. Over a similar dose range of fortification iron added to animal muscle, absorption increased from 0.17 to 3.3 mg iron. These studies indicate that food iron fortification is likely to be effective only in individuals who take animal protein as part of their diet.