Brush border particulates of renal tissue.

Particulates containing a large part of the alkaline phosphatase activity of renal tissue were separated from homogenates and from ribosomal preparations by zonal centrifugation. The particles had a high content of phospholipid and cholesterol that was not removed by treatment with I percent deoxycholate. Enzymatic activities concentrated with the particles were the alkaline phosphatase, a peptidase resistant to proteolysis, glucose-6-phosphatase, inorganic pyrophos-phatase, and adenosine triphosphatase. The particles accumulated leucine with no stimulation from soluble factors and with inhibition by other amino acids; the accumulation was stimulated by adenosine triphosphate and was not inhibited by puromycin. The particles appear to be derived from the membranes of the brush borders of tubular cells.

Potentiation of ifosfamide neurotoxicity, hematotoxicity, and tubular nephrotoxicity by prior cis-diamminedichloroplatinum(II) therapy.

We investigated the relationship between prior therapy and three distinct forms of toxicity that developed during ifosfamide administration (1.6 g/m2/day for 5 days) in 36 children with malignant solid tumors. Of ten therapies that were studied by multiple regression techniques, only the number of doses of cisplatin that patients had received was significantly related to neurotoxicity, hematotoxicity, and tubular nephrotoxicity, with the more severe cases occurring after three or more doses (P less than 0.05). Increased urinary concentrations of the renal tubular enzyme N-acetyl-beta-D-glucosaminidase, measured before each course of ifosfamide, were predictive of neurotoxicity (P = 0.02) and hematotoxicity (P = 0.01). We suggest that cisplatin-induced renal tubular damage, leading to the impaired clearance of ifosfamide metabolites, may account for this added toxicity.

Mesna excretion and ifosfamide nephrotoxicity in children.

To characterize the excretion of 2-mercaptoethanesulfonate sodium (mesna) administered by intermittent infusion, urinary concentrations of mesna and its corresponding inactive disulfide were measured during 50 courses of ifosfamide (1.6 g/m2 for 5 days) and mesna (400 mg/m2 at 0.25, 4, and 6 h after each ifosfamide dose) administered i.v. to 19 patients. Some patients had previously received nephrotoxic therapy that might influence the excretion of mesna and its associated uroprotective effects. The median urinary free thiol concentration increased to 3 mM by 1 h after mesna infusion, declining to background levels by 4 h. The rate of mesna excretion correlated with the creatinine clearance rate in a subset of six patients. The proportion of mesna recovered in urine within 4 h after infusion was lower (P less than 0.05) in children who had evidence of preexisting renal tubular damage. Ifosfamide-induced tubular proteinuria was associated with lower urinary mesna recovery. Low urinary mesna concentrations indicated potentially subtherapeutic renal tubular levels. However, ifosfamide nephrotoxicity was subclinical and is not necessarily linked to differences in mesna excretion.

Urinary N-acetyl-beta-D-glucosaminidase and serum creatinine concentrations predict impaired excretion of methotrexate.

We determined the risk of impaired excretion of methotrexate (MTX) in children with osteosarcoma, who also were receiving cisplatin, by analyzing urinary markers of renal tubular damage, as well as serum creatinine measured before each dose of MTX. MTX clearance was impaired in seven of the ten patients studied after cisplatin therapy. Patients with a urinary N-acetyl-beta-D-glucosaminidase (NAG) concentration of greater than 1.5 U/mmol creatinine or a greater than 50% increase in serum creatinine relative to the pretherapy level were approximately 30 times more likely to have MTX half-lives greater than 3.5 hours than were patients with lower values for these markers; MTX clearance was always impaired if both markers were elevated. If neither urinary NAG nor serum creatinine concentrations increased, the risk of impaired MTX excretion was negligible. Our findings demonstrate that urinary NAG and serum creatinine levels, measured before MTX administration, can be used to identify patients who will have difficulty in clearing the drug.

A rapid means of separating A14-125I-insulin from heterogeneously labeled insulin molecules for biologic studies.

We have used two methods for the preparation of a highly homogeneous insulin with high specific activity. After iodination with chloramine T, the labeled peptides were retained on a disposable Sep Pak cartridge and subsequently eluted. The eluted labeled insulins were further purified by either DEAE cellulose or high performance liquid chromatography (HPLC) to separate A14-125I- from A19-125I-insulin. Both methods of chromatography were effective, but HPLC offered the advantage of better resolution in less time and higher yields of A14-125I-insulin, which is suitable for biologic studies in various target tissues.

Insulin degradation by hepatocytes in primary culture.

The mechanism by which the liver degrades insulin has not yet been completely clarified. In intact, non-"leaky" cells the primary process seems to be mediated by initial receptor binding. We now demonstrate that isolated rat hepatocytes in primary culture are suitable for examining insulin degradation. Hepatocytes did not leak degrading activity into the medium, and thus, the degradation seen was essentially exclusively cell mediated. [125I]Iodoinsulin degradation by these cells was dependent on time and cell concentration. There was a short lag time before degradation products could be detected in the medium. After incubation with the hepatocytes, three peaks of 125I-labeled material could be separated by chromatography on Sephadex G-50. The same three peaks were seen with 125I-labeled material extracted from the cells. When [3H]insulin, labeled exclusively at the B-1 phenylalanine residue, was incubated with the cells, additional peaks of labeled material were recovered from the column. These additional peaks were intermediate in size between insulin and iodotyrosine, suggesting the production of products smaller than insulin but larger than individual amino acids. In order to begin to characterize the subcellular mechanisms for insulin metabolism, the effect of various potential inhibitors on insulin degradation were examined. The most effective inhibitors were N-ethylmaleimide, bacitracin, and Kunitz pancreatic trypsin inhibitor. Chloroquine decreased degradation only 10%, and NH4Cl had no detectable effect. The effect of the inhibitors on the purified insulin-degrading enzyme, insulin protease, was also examined. The purified enzyme responded essentially identically as the intact cells to the various inhibitors. From all these data it would seem that lysosomal degradation of insulin in the hepatocyte may be a relatively minor pathway and the neutral protease may play a major role.

Crustacean defense strategies. I. Molecular weight dependent clearance of dyes in the mud crab Scylla serrata (Forskal) (Portunidae: Brachyura).

Clearance rates of dyes injected into the hemocoel of the mud crab, Scylla serrata, revealed several patterns of clearance and retention associated with physico-chemical and biological variables. Clearance rates remained constant: 1) when the molar concentrations of the injected dyes were equal, 2) after repeated injections of dyes and 3) after serum opsonization. Rates increased: 1) with higher molecular weight irrespective of charge, 2) at higher dye concentration and 3) at night concomitant with an increase in the hemocyte population. In contrast, clearance rates decreased with increasing crab size. Dye cleared from the hemolymph accumulated in the gills but not in the hepatopancreas, antennary glands nor gut. The retention of dyes in the gills increased with higher molecular weights. Granular hemocytes accumulated in the gill rachis and lamellae of dye treated but not in gills of normal and saline injected crabs. A role for hemocytes in molecular weight and concentration dependent clearance of dyes is suggested. Our findings are important for understanding the molecular basis of recognition in crustaceans.

Bone marrow reconstitution of immune responses following irradiation in the leopard frog, Rana pipiens.

The bone marrow of Rana is an important source of cells capable of maintaining individual viability, responding to Concanavalin A (Con A) and producing PFC against sheep erythrocyte (SRBC) antigens. Frog marrow is more effective than the spleen in maintaining life. Radiation destroys the ability of frogs to respond to SRBC immunization (lack of bone marrow and spleen PFC, serum antibody) and bone marrow/spleen cells to respond to Con A, i.e., bone marrow and spleen contain radiation-sensitive cells. Shielding one hind leg during irradiation leads to reconstitution of bone marrow/spleen PFC responses, antibody synthesis and individual viability. Our results suggest that bone marrow is: a) the source of stem cells, and b) the source of mature T- and B- lymphocytes that can recirculate within the immune system.

The very sudden cardiac death syndrome -- a conceptual model for pathologists.

Sudden unexpected death usually involves one of two mechanisms, fast heart stoppage or slow heart stoppage with respiratory arrest. These physiological derangements arise from diverse causes that are sometimes purely functional without structural correlates. The autopsy is considered to be only an investigative tool to be employed and is interpreted in the light of the terminal event, social and medical history, and environmental circumstances.

Monitoring serum aminoglycoside concentrations in children with amphotericin B nephrotoxicity.

We prospectively studied the effect of amphotericin B therapy on aminoglycoside clearance in 20 consecutive children during the remission-induction phase of chemotherapy for acute myelocytic leukemia. Increases (greater than 50%) in the half-life for aminoglycoside excretion were not associated with antileukemic or aminoglycoside therapy alone but occurred in 12 of 17 children when amphotericin B was added to the antimicrobial regimen. Seven children had impaired aminoglycoside clearance without increases (greater than 50%) in serum creatinine; hence the resulting adjustments in aminoglycoside dosage would not have been made had we relied solely on serial measurements of serum creatinine. Evidence for increased excretion of the renal enzymes N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase during amphotericin B therapy suggested that damage to proximal tubular cells may contribute to the renal impairment that has been associated with this drug. Our findings underscore the value of monitoring serum aminoglycoside concentrations in children being treated with amphotericin B.

Cancer chemotherapy-induced tubular nephrotoxicity evaluated by immunochemical determination of urinary adenosine deaminase binding protein.

The authors evaluated measurements of adenosine deaminase binding protein (ADB), a proximal renal tubular cell antigen, for detection of drug-induced tubular nephrotoxicity. Concentrations of ADB were determined immunochemically in serial urine specimens from 12 children who were receiving chemotherapy for malignant solid tumors. There was no indication of increased ADB excretion after administration of two nonnephrotoxic drugs, etoposide and doxorubicin, but in patients given the recognized nephrotoxins, cisplatin and methotrexate, or an investigational drug, ifosfamide, urinary concentrations of ADB increased greater than fivefold relative to baseline values. Increased ADB concentrations preceded cisplatin- or ifosfamide-induced elevations of serum creatinine. Results of the ADB assay correlated well with those obtained by enzymatic assays for N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase (r = 0.76 and 0.53; n = 142, P less than 0.001) and marginally with total proteinuria (r = 0.21; P less than 0.02). Hence, serial ADB measurements may be useful in screening investigational drugs for acute subclinical nephrotoxicity.

Cumulative renal tubular damage associated with cisplatin nephrotoxicity.

We assessed the acute and chronic effect of multiple courses of cisplatin therapy on renal tubules by monitoring the urinary excretion of alanine aminopeptidase, N-acetyl-beta-D-glucosaminidase, and total protein. Urine specimens were obtained before and after doses of cisplatin (90 mg/m2) given to 12 patients. Each dose of cisplatin induced transient increases in enzyme excretion, followed by proteinuria 3-5 days later. Transient enzymuria after the last cisplatin dose was significantly greater than that after the first dose. Moreover, persistent increases in urinary N-acetyl-beta-D-glucosaminidase and serum creatinine concentrations over pretherapy levels indicated chronic renal tubular damage. Our findings disclosed striking differences between patients in susceptibility to progressive nephrotoxicity.

Carboplatin (CBDCA), iproplatin (CHIP), and high dose cisplatin in hypertonic saline evaluated for tubular nephrotoxicity.

We compared the acute tubular nephrotoxicity of three platinum compounds in children and adults with solid tumors by monitoring the urinary excretion of alanine aminopeptidase, N-acetyl-beta-D-glucosaminidase, and total protein. Cisplatin (100 mg/m2) was administered with mannitol, or at a twofold larger total dosage (50 mg/m2 per day for 4 days) in a 3% saline infusion. Carboplatin (300 mg/m2) was administered in combination with 5-fluorouracil, and iproplatin was administered in dosages ranging from 216 to 388 mg/m2. Enzymuria and proteinuria induced by cisplatin at a total dosage of 200 mg/m2 on a divided schedule did not significantly differ from that observed for the single 100 mg/m2 dose. Enzymuria and proteinuria induced by carboplatin and iproplatin were significantly less than that for cisplatin; however, one patient developed chronic tubular damage after three courses of carboplatin, and the acute tubular toxicity of iproplatin in one of 15 patients was exceptional. Our findings support the value of administering cisplatin in hypertonic saline on a divided schedule as a strategy to reduce acute tubular damage. Although carboplatin and iproplatin are less nephrotoxic than cisplatin, occasionally patients experience subclinical acute or chronic tubular damage that may lead to overt nephrotoxicity with continued therapy.

Increased levels of urinary adenosine deaminase binding protein in children treated with cisplatin or methotrexate.

Levels of adenosine deaminase binding protein (ABP), a renal tubular cell antigen, were determined by enzyme immunoassay in urine specimens from seven children with solid tumors who were receiving the recognized nephrotoxins cisplatin or methotrexate. ABP excretion was uniformly increased within the first 3 days after administration of either drug. Elevated ABP levels were usually accompanied by increased excretion of the urinary enzymes N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase. In alkaline urine specimens associated with methotrexate therapy, ABP levels were increased whereas enzyme activities appeared to be unstable. Hence, immunochemical measurement of urinary ABP levels may be adjunctively useful for clinical studies of renal tubular damage.

Death or injury caused by explosion.

Explosions may have varied energy sources, reasons for occurring, and ways of injuring or killing. Careful analysis of the explosion scene, coupled with an equally careful examination of surgical or autopsy material as the result of an explosion, contributes greatly to the investigation. Surgical or autopsy material suspected of having been injured by explosion should be carefully photographed and x-rayed, swabbed with chloroform-soaked swabs, then swabs soaked in 0.1N hydrochloric acid, cleaned meticulously while collecting only surface fragments, photographed and x-rayed again, and then searched extensively for fragments.

Death or injury caused by electrocution.

Proper investigation of injury and death from electrocution requires a high level of suspicion, as examination of the victim will often prove negative. Careful photographic documentation of the scene must be done in every case. In low voltage cases, the equipment that may have been involved should be photographed, x-rayed, and examined electrically. Autopsy examination of the victim in cases of electrocution due to high voltage alternating or direct current usually reveals burns and the nonspecific findings of asphyxia. Victims of low voltage alternating current often have no electrical burns and the absence of findings characteristic of ventricular fibrillation. Low voltage direct current rarely produces death.

Sudden death due to ingestion of cocaine.

A 27-year-old male died as the result of swallowing a bag of cocaine. Using gas chromatography and ultraviolet spectrophotometry, the concentration of cocaine was determined on all specimens submitted for analysis. These analyses revealed what may possibly be the highest concentrations of cocaine ever reported in the toxicology literature. These values were confirmed by another medical examiner toxicology laboratory.

The investigation of electrical deaths: a report of 220 fatalities.

We present the results of our investigation of 220 electrocutions. The ratio of high voltage to low voltage electrocution is found to be nearly 1:1. Further, in low voltage electrocution deaths electrical burns were absent in over 40% of the cases. Our approach to the investigation of possible electrocution, including equipment analysis, is discussed.

Fatal virus-associated hemophagocytic syndrome in a young adult producing nontraumatic splenic rupture.

A 24-year-old man with no previous medical history was admitted to a local hospital with pancytopenia after a recent "viral illness." During his hospitalization, he developed sudden abdominal distension and hypotension. Surgical exploration of his abdomen revealed a ruptured spleen. The spleen was removed, but the patient did not survive the operation. We investigated this unexpected and unexplained hospital death for any traumatic or iatrogenic injury. The cause of death after review of the clinical history, autopsy, and microscopic sections was virus-associated hemophagocytic syndrome (VAHS). VAHS consists of a generalized histiocytic proliferation and marked hemophagocytosis associated with a systemic viral infection. Clinically it presents as pancytopenia and organomegaly. This recently described entity is often confused with malignant histiocytosis. This is the first case report of VAHS producing nontraumatic splenic rupture, thus adding to the differential diagnosis of spontaneous splenic rupture and sudden natural death.