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Red-tailed hawks were exposed to sublethal levels of lead acetate for periods of 3 or 11 weeks. Alterations in the heme biosynthetic pathway were demonstrated after the first week of exposure to 0.82 mg lead per kilogram body weight per day. Activity of erythrocyte porphobilinogen synthase (aminolevulinic acid dehydratase) was depressed significantly and did not return to normal levels until 5 weeks after the termination of lead treatments. A rapid and relatively brief increase in erythrocyte free protoporphyrin and a slower but more prolonged increase in its zinc complex were also demonstrated with exposure to this dose of lead for 3 weeks. Less substantial decreases in hematocrit and hemoglobin levels occurred but only in the longer experiment with exposure to higher lead levels. Short term, low level lead exposure did not effect immune function significantly in the hawks, as measured by antibody titers to foreign red blood cells or by the mitogenic stimulation of T-lymphocytes. Increased lead exposure produced a significant decrease in the mitogenic response but had no effect on antibody titers.

Osteosarcoma is the most common malignant bone tumor in dogs and, like its human orthologue, is characterized by aggressive local behavior and high metastatic rates. The Scottish deerhound is a breed of dog with a >15% incidence of osteosarcoma and represents an excellent spontaneously occurring large-animal model of the human disease. We modeled the transmission of the osteosarcoma phenotype in a population of over 1000 related deerhounds ascertained as part of a prospective health study. Variance component analysis, segregation analysis, and linear modeling were performed to evaluate heritability, to infer the presumptive transmission model, and to identify covariate effects for this phenotype within the breed, respectively. Based on variance component analysis, heritability (h2) was estimated to be 0.69. Six transmission models were analyzed by segregation analysis; based on Akaike's information criteria, the most parsimonious model was the Mendelian major gene model with dominant expression. Linear modeling identified gender and genotype as significant predictors of disease outcome. Importantly, duration of gonadal hormone exposure, weight, and height at maturity were not significant predictors of outcome. Inheritance of the putative high-risk allele was thus associated with >75% risk of disease occurrence compared to the

As a subject's true disease status is seldom known with certainty, it is necessary to compare the performance of new diagnostic tests with those of a currently accepted but imperfect 'gold standard'. Errors made by the gold standard mean that the sensitivity and specificity calculated for the new test are biased, and do not correctly estimate the new method's sensitivity and specificity. The traditional approach to this problem was 'discrepant resolution', in which the subjects for whom the two methods disagreed were subjected to a third 'resolver' test. Recent work has pointed out that this does not automatically solve the problem. A sounder approach goes beyond the discordant test results and tests at least some of the subjects with concordant results with the resolver also. This leaves some issues unresolved. One is the basic question of the direction of biases in various estimators. We point out that this question does not have a simple universal answer. Another issue, if one is to test a sample of the subjects with concordant results rather than all cases, is how to compute estimates and standard errors of the measures of test performance, notably sensitivity and specificity of the test method relative to the resolver. Expressions for these standard errors are given and illustrated with a numeric example. It is shown that using just a sample of subjects with concordant results may lead to great savings in assays. The design issue of how many concordant cells to test depends on the numbers of concordants and discordants. The formulae given show how to evaluate impact of different choices for these numbers and hence settle on a design that gives the required precision of estimates.

Protoporphyria, a photosensitizing disease documented only in humans, was transmitted as a recessive trait to seven female calves. Cutaneous lesions were extensive, and erythrocyte and fecal protoporphyrin concentrations exceeded by far those of human protoporphyria. Average ferrochelatase activity was decreased to one-half of normal in the liver of carriers, and to about one-tenth of normal in liver, kidney, heart, spleen, lung, and marrow of protoporphyrics.

1. 1. Two new procedures are described for the quantitative assay of “free” and zinc-protoporphyrin in red blood cells. 2. 2. Major clinical findings have included the following: (i) An average of about 10% of the total erythrocyte protoporphyrin is “free” in normal humans, but about one-third or more is usually “free” in blood of normal cows, rats and rabbits, (ii) In addition to human and bovine protoporphyria, “free” protoporphyrin was increased in (a) genetic carriers (clinically normal) of bovine protoporphyria, (b) acute Pb poisoning, and (c) some patients undergoing dialysis, (iii) Significant amounts of zinc-coproporphyrin were found in the blood of several dialyzed patients and in some “normal” cows.

The only type of hereditary porphyria described in nonhuman species is the rare erythropoietic (“congenital,” or “Gunther1s”) type. Hereditary bovine protoporphyria, which exhibits an acute photosensitivity even during the 1st week of life, has now been documented (3) in four calves sired by the same clinically normal bull. Their normal-appearing dams were genetically related to this bull. Teeth and urine were normal in color, and had no red fluorescence on exposure to nearly UV light. Concentrations of protoporphyrin in red cells and feces were higher by far than any reported in human protoporphyria; both exceeded 35,000 yg/100 ml (g). Fecal urobilinogen (u’gen) was markedly increased. Ferrochelatase activity was decreased to less than 15% of normal in kidney, spleen, lung, and marrow. It was also decreased in cultured skin fibroblasts (Sassa, unpublished studies), but was approximately half normal in the liver of each of four carrier dams. ALA synthase activity (B. Schacter) was increased slightly, and only in the liver of protoporphyrics. These and other findings are summarized in Table 1. In animals removed from pasture to a dimly lit barn, fecal protoporphyrin and urobilinogen values rapidly approached normal and skin lesions healed, but red cell porphyrin remained above 35,000 yg/100 ml.

[Porphyrin-14C] cytochrome c (isolated from tissues of dogs injected with [14C] ALA) was given intravenously to one normal and one bile fistula dog. Essentially all of the injected 14C was excreted in the urine during the first six days. No (unchanged) cytochrome c was detectable in the urine. Analysis of 14C and of light absorption at 400 nm in the successive eluates from Florisil columns showed that all 14C peaks coincided with pigment peaks, but some pigment peaks has no increase in 14C. The relative distribution of 14C in these pigment peaks changed markedly between the first and third days. Delayed excretion of some 14C suggested cellular uptake of cytochrome c prior to the urinary excretion of its endogenous metabolites.

We describe the use of Florisil chromatography to purify and fractionate porphyrins into uro-, copro-, and protoporphyrins before their measurement by quantitative fluorometry. Porphyrinogens in fresh urine may be rapidly oxidized to porphyrins by chloranil, or slowly in weakly alkaline solution, before the sample is applied to the Florisil column. Aliquots of the separate ammoniacal eluates may be lyophilized for additional fractionation by direct thin-layer chromatographic analysis if desired. The behavior of delta-aminolevulinic acid, porphobilinogen, and other pyrrole compounds in this system is also described.

A population of 163 incarcerated delinquents (126 males and 37 females aged 12–18 years) was studied to determine the prevalence of iron deficiency and to compare hemoglobin (Hgb), mean corpuscular volume (MCV), serum ferritin (SF), and erythrocyte protoparphyrin/hemoglobin (EP/Hgb) as predictors of response to iron therapy. Thirty-two percent of females and 6% of males had SF ≤ 12 ng/ml; 51% of females and 24% of males had SF ≤ 20 ng/ml. The mean SF was 17.7 ng/ml for females and 29.2 ng/ml for males. Of the 163 subjects, 53 were at risk for iron deficiency based on SF, Hgb, EP/Hgb, or MCV criteria. Twenty-one completed treatment with iron, and nine had greater than 1 g rise in Hgb. The following tests identified responders: SF ≤ 12 ng/ml-5/9; SF ≤ 20 ng/ml-9/9; Hgb ≤ third percentile-4/9; Hgb ≤ tenth percentile-7/9; MCV ≤ tenth percentile-2/9; EP/Hgb ≥ 3.0 μg/g Hgb-2/9; EP/Hgb ≥ 2.5 μg/g Hgb-4/9. Serum ferritin ≤ 20 ng/ml had a false positive rate of 57%; Hgb ≤ tenth percentile and EP/Hgb ≥ 2.5 μ g/g Hgb had no false positives. The significance of the high prevalence of iron deficiency among a population of incarcerated adolescents is not clear. Serum ferritin ≤ 20 ng/ml offers the most sensitive screening test to suggest iron-responsive anemia, but it has poor specificity; Hgb ≤ tenth percentile is an alternative screening test that is more available, less expensive, nearly as sensitive, and more specific; EP/Hgb ≥ 2.5 μg/g Hgb, MCV ≤ tenth percentile, SF ≤ 12 ng/ml, and Hgb ≤ third percentile did not prove to be good screening tests because of poor sensitivity.

889 ERYTHROCYTE PROTOPORPHYRIN (EP): THE RATIO OF FREE EP TO ZINC-COMPLEXED EP IN NORMAL CHILDREN

EP exists in both the free state (FEP) and in a zinc complexed form (ZEP) in RBC. The ratio of FEP to ZEP in healthy individuals averages 1:10. In the newborn period and in conditions with increased RBC turnover the FEP/EP ratio becomes elevated. Using dextran or stractan gradients, cord blood was centrifugally separated into 4 fractions known to contain RBC populations of different ages. The characteristics of these fractions and their EP contents are as follows: (N=6, EP expressed as μg/dl RBC). The total content of EP did not vary significantly according to RBC age but there was an increased FEP/EP in the fraction of younger, reticulocyte-rich cells. This EP pattern suggests that the newly formed EP is in the free state and may become zinc complexed or lost from RBC with time. The previous observation of an elevation in the free form of EP in hemolytic disorders is likely to be related to the presence of a larger percent of young RBC.