Samuel Schwartz

A Simple Test for Urinary Porphobilinogen.

The urines of patients suffering from idiopathic porphyria have often been noted to exhibit strong Ehrlich reactions, in many instances at least, not due to urobilinogen. Waldenströms studies 1 , 2 clearly demonstrated that the chromogen responsible for the Ehrlich reaction in these urines is quite distinct from urobilinogen, which is of course, most often implicated in other pathological states. During the past 3 years we have had opportunity to investigate urine samples from 5 cases of so-called “acute” idiopathic porphyria.† In each instance the urine contained the zinc complex of uroporphyrin in considerable amount. The subject of zinc uroporphyria as a disease entity will be considered in a separate communication. These urines also exhibited Ehrlich reactions in varying degree, at times very intense. In the first 4 cases, the freshly voided urine was already red-brown in color, becoming darker on exposure to light. In the fifth case, the fresh urine was for the most part normal in color, exhibiting a strong Ehrlich reaction. On standing in the light for several days the urine from this case likewise became a deep reddish-brown, containing now both the zinc complex of uroporphyrin and the reddish-brown porphobilin. 2 Waldenström pointed out that porphobilinogen is much less soluble in organic solvents than is urobilinogen. 1 , 2 In addition to confirming this, we have found that the porphobilinogen aldehyde compound as formed in the Ehrlich reaction is wholly insoluble in chloroform, while that of urobilinogen is readily and easily extracted with this solvent. The reaction as we have carried it out is as follows: Equal parts of urine and Ehrlichs reagent‡ are mixed in a test tube.

Fecal blood levels in health and disease. A study using HemoQuant.

We tested HemoQuant, a quantitative assay of fecal blood based on the fluorescence of heme-derived porphyrin, in 106 healthy volunteers, 170 patients with gastrointestinal symptoms but with normal diagnostic studies, 44 patients with gastrointestinal cancer, 75 patients with benign polyps, and 374 patients with a variety of other benign gastrointestinal lesions, including ulcers and erosions. In 98 per cent of the healthy volunteers, fecal hemoglobin concentrations were less than 2 mg per gram of stool. Levels were similarly low in stools from patients with symptoms and normal studies and in patients with relatively minor benign lesions. Within these groups, levels were slightly higher in those who had ingested red meat or aspirin. The fecal hemoglobin concentration was higher in patients with gastrointestinal cancer than in any other group, and 97 per cent of those with colorectal cancer had levels above 2 mg per gram. The sensitivity of HemoQuant was significantly greater than that of the guaiac test Hemoccult, particularly when heme was degraded or stools were dry. Intestinal degradation of heme to porphyrin can be measured separately by HemoQuant, and was greater when bleeding was from proximal lesions rather than distal ones. We conclude that HemoQuant is a more sensitive measure of gastrointestinal bleeding than Hemoccult, and that its capacity to measure degraded heme may be useful in indicating the anatomic site of bleeding.

Gastrointestinal Blood Loss and Anemia in Runners

Iron deficiency, with or without anemia, occurs commonly in long-distance runners, but the cause is unknown. The recent development of a simple quantitative assay for fecal hemoglobin, HemoQuant , allowed us to study whether gastrointestinal bleeding occurs in runners. Blood and stool samples were collected from 24 runners before and after a race of 10 to 42.2 km and from age- and sex-matched, nonrunning controls. The mean blood hemoglobin level and hematocrit were significantly lower in runners than in controls. Serum ferritin levels were below normal in 4 runners but in no controls. Fecal hemoglobin levels increased in 20 of 24 runners (p less than 0.01) after a race. Mean fecal hemoglobin level was 1.08 mg/g (range, 0.11 to 2.36) in controls and 0.99 mg/g (0.18 to 2.41) in runners before a race, but peaked at 3.96 mg/g (0.37 to 43.20) after a race. Competitive long-distance running induces gastrointestinal blood loss and may contribute to iron deficiency.

Experimental Porphyria. III. Hepatic Type Produced by Sedormid.

Summary and Conclusions 1. Allylisopro-pylacetylcarbamid (“Sedormid”) produces an hepatic form of porphyria in rabbits. 2. The livers of these animals contain large amounts of proto-, copro- and uroporphyrin, the latter chiefly in the form of non fluorescing precursors. Porphobilinogen is also present. 3. The protoporphyrin is excreted only, and the coproporphyrin chiefly, in the bile, most of the uro- type porphyrins appearing in the urine, in amounts ranging up to 60 mg in 24 hours. The latter consists mainly of type III isomers, but type I has also been identified in small amount. Large amounts of porphobilinogen are excreted in the urine. 4. The porphyrin content of erythrocytes, bone marrow, spleen, and brain is within normal limits. 5. Transient paresis of hind limbs, dilatation of the stomach, and constriction of the pylorus are noted, with irregular small bowel spasm. Death is invariably due to rupture of the stomach. 6. The possibility is discussed that the genesis of the porphyria is related to a primary effect of the Sedormid on the formation of iron porphyrin enzymes in the liver cell.

Effects of chronic exposure to sublethal concentrations of lead acetate on heme synthesis and immune function in red-tailed hawks.

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.

Quantitative fecal recovery of ingested hemoglobin-heme in blood: Comparisons by HemoQuant assay with ingested meat and fish

Blood, meat, or fish, or any combination thereof, were ingested by 9 normal volunteers to permit studies of the contained hemes during total gastrointestinal transit. Quantitative analysis of ingested heme and of fecal heme and its degradation products was made possible by a new specific and extremely sensitive test, HemoQuant. The average fecal recovery of hemoglobin-heme from 10 to 36 ml of blood was 88%, as determined in 13 separate studies. All Hemoccult tests remained negative despite greater than 20-fold increases in fecal heme. Up to 83% of the blood heme was converted in the intestinal tract to porphyrins. These porphyrins are included in the HemoQuant, but not in Hemoccult or other leukodye assays. Negligible amounts of heme were found in fish and fowl, and their ingestion led to no significant increase in fecal heme. An average of only 25% of the heme in ingested meat was subsequently recovered in feces. Control fecal values represented an average of approximately 0.5 ml of blood per day. The recovery data obtained show that fecal HemoQuant results reliably reflect the total amount of blood hemoglobin that enters the gastrointestinal tract.

A Stool Collection Device: The First Step in Occult Blood Testing

Despite widespread fecal blood testing, the technique of gathering stool for sampling has remained uncontrolled. We sought to describe how patients have contended with this awkward step, to study artifact caused by toilet water, and to construct a collection device that prevents sampling problems. A survey of 250 patients showed that most (56%) had retrieved stools from the toilet basin, 17% used a pan or other household receptacle, 10% used newspaper or tissue paper, and 17% had been unable or unwilling. Sampling stool from the toilet basin introduces error because 4% to 75% of blood leaches from the fecal surface into surrounding water after only 4 to 12 minutes, and many toilet sanitizers cause false-positive guaiac reactions. We describe an inexpensive, disposable stool collector; outpatient compliance has been 97% using this device. To avoid biochemical artifact and facilitate stool sampling, we advocate that a collection device be incorporated into the occult blood testing process.

Quantitative assay of erythrocyte “free” and zinc-protoporphyrin: Clinical and genetic studies

Abstract 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.

Green jaundice. A study of serum biliverdin, mesobiliverdin and other green pigments.

Green jaundice is generally ascribed to biliverdinemia, but no proof of the identity of responsible pigments is known. A serum biliverdin concentration of 3 mg%, found in 1 of our patients, is thought to be the highest such value recorded. The accepted analytic method employed, however, was found to be nonspecific; a mixture of biliverdin, mesobiliverdin and related pigments appeared to contribute to the green color observed. The use of Triton X-100 and KCN facilitated spectrophotometric distinction of these pigments from green hemoprotein in the 3 illustrative patients described.

Early labeling of bilirubin from glycine and δ-aminolevulinic acid in bile fistula dogs, with special reference to stimulated versus suppressed erythropoiesis

Abstract Incorporation of glycine-2-C 14 into Hb proto and bile bilirubin was studied at short intervals in 2 bile fistula dogs during successive periods of inhibited and then stimulated erythropoiesis (plethora and bleeding in 1 dog and undefined illness and bleeding in another), and in a third dog with bile renal fistula but without disturbed erythropoiesis. Results support the concept of a nonerythropoietic component as a partial source of early-labeled bilirubin. Thus, during the period of inhibited erythropoiesis relatively larger amounts of bilirubin-C 14 were excreted during the first 48 hours. The total amount of this labeling was appreciably greater than that expected from the relatively low labeling of total circulating hemoglobinproto during this period compared to the postbleeding period. The per cent incorporation of glycine-C 14 in total circulating Hb proto during the postbleeding and plethoric periods, respectively, were 5.8 and 0.48 (ratio of 12.1), while the corresponding values for the total 6 day bile bilirubin-C 14 were 0.22 and 0.04 (ratio of 5.5). In an otherwise normal bile fistula dog given ALA-C 14 , 20 per cent of the C 14 administered was recovered as bile bilirubin-C 14 during the following 6 days, with 94 per cent of this amount appearing during the first 2 days. Peak activity at 3–4 hours was 22 × 10 6 dpm/mg./mc. dose. Only 0.26 per cent of the dose was incorporated in circulating Hb proto in contrast to 3.0 per cent in the normal dog given glycine-C 14 . In a second dog studied for only 24 hours, 19.5 per cent of the administered dose of ALA-C 14 was excreted as bile bilirubin-C 14 .