Membrane protein pattern in hereditary spherocytosis in five subjects from north-east Italy obtained by SDS-PAGE using N,N'-diallyltartardiamide.

In the present study we examined five subjects affected by hereditary spherocytosis (three unsplenectomized and two splenectomized), coming from an area in the north-east of Italy where hereditary spherocytosis is an anaemic disease with very low incidence. All patients showed a low degree of spectrin deficiency (14%), detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, when this analysis was performed with N,N'-diallyltartardiamide as cross-linking agent instead of N,N'-methylenbisacrylamide, some unusual bands appeared in the region between proteins 4.2 and 5, the three unsplenectomized and two splenectomized patients showing different patterns. We hypothesise that some alterations of proteins in this region (e.g. the 4.5 or 4.9 bands), possibly due to proteolysis, must have occurred in relation to the disease.

An easy diagnostic approach to primary aldosteronism.

The infusion of 40 mEq potassium (aspartate) in 250 ml isotonic 1-fructose at a rate of 20 mEq/h into 5 patients (34-56 years old) with aldosteronoma and 2 patients with bilateral primary aldosteronism consistently raised their mean arterial pressure by 15-20 mmHg. Their pressure values returned to the baseline levels 4-5 h after the infusion. In contrast, in controls (10 patients with idiopathic arterial hypertension, matched for age, sex, and magnitude of the untreated hypertension, and 7 patients with inactive adrenal nodules as incidental findings on upper abdomen ultrasound or computerized tomography) the same procedure caused negligible arterial pressure changes. The cause of the rise in blood pressure observed uniquely in patients with primary aldosteronism after infusion of potassium (aspartate) cannot be accounted for by an increase in plasma aldosterone, blood volume, or plasma angiotensin II. The cause of this response thus remains obscure; nonetheless, this simple procedure may prove useful in differentiating primary aldosteronism from idiopathic hypertension, in excluding the adrenal disorder, and in revealing even its mildest forms.

Neuron-specific enolase, thymidine kinase, and tissue polypeptide-specific antigen in diagnosis and response to chemotherapy of small-cell lung cancer.

The clinical usefulness of neuron-specific enolase (NSE), thymidine kinase (TK), and tissue polypeptide-specific antigen (TPS) was investigated in 41 patients (53-80 years old) with recently discovered small-cell lung cancer (SCLC). Eleven patients exhibited limited disease (LD) and 30 extensive disease (ED). Serum samples for NSE, TPS (immunoradiometric assay), and TK (radioenzymatic assay) evaluations were drawn from all patients at the time of diagnosis and before each cycle of chemotherapy in the treated patients. Therapy consisted of i.v. carboplatin 300 mg/m2 on the first day and i.v. etoposide 120 mg/m2 from the first to the third day every 3 weeks. Nine patients refused or were not eligible for chemotherapy. Five patients received only one course and showed no response (NR); 9 patients received two courses; 18 patients received three or more courses. In the last group, complete remission (CR) was obtained in 9 cases, partial remission (PR) in 18 cases. The tumor markers studied did not show any significant difference in distinguishing LD from ED. NSE and TPS were significantly more often abnormal than TK, either at the time of diagnosis (p < 0.05) or in PR or NR patients (p < 0.05). In relation to chemotherapy response, NSE and TPS serum patterns were shown to be more reliable than TK in PR (p < 0.05) and NR patients (computed error between 10% and 15%). No significant difference was observed between serum NSE and TPS patterns. Serum NSE and TPS seem to be more useful in the diagnosis and follow-up of SCLC patients undergoing chemotherapy. Further trials are necessary to ascertain whether the associated assessment of NSE and TPS can add useful information to that provided by the assessment of NSE alone.

Platelet activation and fibrinolysis in large bowel cancer.

Four coagulation indices [fibrinopeptide A (FpA), X degradation products (XDPs), platelet factor (PF4), beta-thromboglobulin (beta-TG)] were assessed in 38 patients affected by large bowel cancer at different stages before surgery, 7 and 30 days after it, to evaluate the capacity of such neoplasia to influence coagulation. The reported study showed that (1) FpA levels were elevated in nearly all cases both before and after surgery (thus it is a useful index for the diagnosis of such neoplasia, although its levels are not influenced by the presence of metastases), (2) increased levels of XDPs were found in a significantly (p < 0.05) higher percent of patients 7 days after surgery (no significant differences were observed in relation to the cancer stage), (3) PF4 levels were in the normal range throughout the study, and (4) beta-TG levels were increased throughout the study in a high percentage of cases. It may be inferred that large bowel cancers may affect coagulation in either way(s), by triggering platelet activation and/or fibrinolysis. The most useful index for the diagnosis and follow-up of this neoplasia is FpA, although its levels are unaffected by the presence of metastases. The comprehensive evaluation of the four above-mentioned coagulation indices may give rise to the suspicion of large bowel cancer.

Beneficial effects of verapamil in renal-risk surgical patients.

OBJECTIVE: To test whether the administration of calcium channel antagonists, such as verapamil (V), on the day before, during, and for 24-36 h after an important abdominal intervention, can lower the onset of acute renal failure (ARF), mostly in renal-risk patients, such as the aged. DESIGN: Randomized, nonblinded study. SETTING: Three surgical care university departments and two intensive care units of the same hospital (S. Anna, Ferrara, Italy). PATIENTS: Thirty-five elderly patients (61-83 years old) entered the study: 18 of them were given V; 17 were not treated and were considered as controls. The two study groups were overlapping as regards age, renal risk, and surgical challenge. The patients who underwent ARF (5 in the treated group, 7 among the controls) were rejected from the study. INTERVENTIONS: V was given on the eve of surgery at a dose of 80 mg/8 h per os and then through slow infusion (5 mg/4-6 h) during the next intra- and postoperative 24-36 h. Abdominal surgery was performed owing to gastric cancer (8 cases), colorectal neoplasia (10 cases), gallstone disease (4 cases), subrenal aortic aneurysm (6 cases), and iliofemoral obstructive arteriopathy (7 cases). MEASUREMENTS: Serum creatinine (SCr) was assessed to test renal function; 24-h urinary levels of brush-border enzymes (gamma glutamyl transferase, or gGT), lysosomal enzymes (N-acetyl-beta-D-glucosaminidase, or NAG), and beta 2-microglobulin (or beta 2M) were determined at T0 (on the eve of surgery), T1 (first and second day after), and T2 (7th and 8th day after) to demonstrate possible tubule cell damage. RESULTS: In the evaluated patients (13 treated with V and 10 untreated): (a) the 24-h urinary levels of gGt and NAG persisted unchanged throughout the study in the treated patients, whereas in the controls the same indices exhibited significant (p < 0.01) increases at T1 and T2; (b) the 24-h urinary levels of beta 2M showed significant (p < 0.01) increases in both groups from T0 to T1; however, at T2 these values tended to return to normal ranges in the treated patients, whereas they continued to be elevated in the untreated group. As regards the patients who underwent postoperative ARF, in the treated group urine output was significantly larger (p < 0.01 at T1 and p < 0.001 at T2), SCr was significantly (p < 0.05) lower, and the renal function recovered earlier (within 10 +/- 3 vs. 22 +/- 9 days) than in the controls. CONCLUSIONS: The administration of calcium channel antagonists to renal-risk patients during surgery and immediately before and after it has failed to prevent the onset of postoperative ARF. Nevertheless this procedure has been shown to somehow reduce surgery-mediated lesions of the tubule cells, as demonstrated by the finding of elevated urinary enzymes only in the untreated group.

The importance of the highest normokalemia in the treatment of early hepatic encephalopathy.

An inverse relation is known to link blood potassium with renal synthesis and the release of ammonia. Given the liability of hyperammonemia for precipitating hepatic encephalopathy (HE), 28 patients affected by stage I HE were equally divided into two groups and maintained up to their death at the highest (5.4-5.5 mEq/l) or the lowest (3.5-3.6 mEq/l) normokalemia levels. When compared with the lowest normokalemia group, the highest one showed an early, albeit transient, improvement in the mental state (as assessed by both EEG and psychiatric investigations) and to a lesser extent in hepatic functions (as assessed by the variations in serum bilirubin, GPT, GGT and plasma prothrombin time). In the highest normokalemia group the survival was also prolonged. The cause of this improvement may be related to the induced decrease in blood pH, the consequent depression of renal ammoniagenesis and the rise in the arterial and urine NH+4/NH3 ratios. These factors reduce the entry of ammonia into the cells and enhance the urinary excretion of this metabolite, respectively.

Usefulness of the assessment of urinary enzymes and microproteins in monitoring ciclosporin nephrotoxicity.

The clinical usefulness of serial assays of urinary N-acetyl-beta-D- glucosaminidase (NAG), gamma-glutamyltransferase (GGT) and beta 2-microglobulin (beta 2M) were tested to evaluate and follow up the nephrotoxicity resulting from the prolonged administration of ciclosporin (CS). Three groups of patients were studied for 18 months: group A: functioning renal transplant patients (n = 13) on maintenance therapy from 12-31 months with CS and prednisone; group B: functioning renal transplant patients (n = 11) treated with prednisone and azathioprine; group C: patients (n = 10) affected by autoimmune steroid-unsensitive uveitis, free from previous renal disorder and treated with CS (for 8-16 months) at progressively decreasing doses. In groups A and B, the urinary enzymes and beta 2M underwent overlapping increases, so that these parameters cannot be considered reliable indices of CS-induced nephrotoxicity. This is due to the fact that transplanted kidneys are already altered by concomitant or preexisting affections, or by persistent immunologic injury. Conversely, in patients with uveitis, the serial assays of such urinary parameters prove to be quite reliable to evidence clinically yet unrecognizable kidney involvement due to prolonged CS administration. High enzymuria has been shown to be an earlier marker of nephrotoxicity only in nephropathy-free patients; on the other hand, the regression of elevated beta 2Muria into normal ranges indicates complete tubule cell recovery.

Coagulation disorders and tumor markers in the diagnosis of pancreatic cancer.

Twenty patients (42-80 years old of whom 9 women) affected by instrumentally ascertained pancreatic cancer (7 cases were operated on) were studied. In all of them the following coagulation indices (fibrinopeptide A, FpA; beta-thromboglobulin, BTG; platelet factor IV, PF4; fibrinogen degradation products, XDP) and tumor markers (gastrointestinal cancer associated antigen, GICA; tissue polypeptide antigen, TPA; carcinoembryonic antigen, CEA; alpha-fetoprotein, or AFP) were assessed at the time of diagnosis, and 10 and 30 days after diagnosis, to test whether and which of the above parameters are more sensitive for entertaining the underlying affection. In both operated and nonoperated patients FpA was shown to be the most sensitive index. Lesser sensitivity was shown by XDP, GICA, and BTG. AFP proved to be quite useless as its serum levels constantly fell within the normal range.

Specificity of tumor markers (CEA, GICA, TPA, alpha-FP, FpA, gamma-GT) for the diagnosis of hepatic metastases from large bowel cancers.

In 98 patients affected by colorectal cancer (43 patients with colon cancer, 55 patients with rectosigmoid cancer) the specificity of some tumor markers (CEA, GICA, TPA, alpha-FP, FpA, gamma-GT) has been tested in evidencing the coexistence of liver metastases and the site of the primary tumor, i.e. the rectosigmoid region (rectum + 15 cm of the adjacent sigmoid colon) vs the rest of the colon. Liver metastases, present in 19 patients with colon cancer and in 24 with recto-sigmoid cancer, were previously ascertained by various instrumental investigations. Unlike previous studies which indicated CEA or alpha-FP as the most reliable markers to suggest the coexistence of liver metastases in such patients, the reported results allow the following sequence, in decreasing order of sensitivity, to be proposed: gamma-GT; FpA; CEA and GICA to a similar degree; TPA, which increases only when liver metastases from colon cancer are present; lastly, alpha-FP, which rises only in very few cases of massive hepatic involvement.

Different kalemia in abdominal trauma.

Unlike previous reports, in 123 patients with abdominal injury or surgery significant and persistent hypokalemia was observed only after traumatic or surgical injuries of the liver. On the contrary, in all other extrahepatic abdominal trauma or operations kalemia was normal. This discrepancy may be related to the much greater abundance, in comparison with other abdominal organs, of adrenergic endings present in the liver. Trauma to or surgery of the liver stimulates such fibers, which locally release proportionally large amounts of norepinephrine. The consequent stimulation of beta-adrenergic receptors enhances the transfer of potassium from the blood into the cells, even if the affinity of norepinephrine for such receptors is less than that of epinephrine. The frequent finding of kaliuria at the upper normal levels may suggest an accessory role of aldosterone in causing and maintaining this hypokalemia.

Life-threatening hyponatremia caused by vinblastine.

Some days after the administration of a third bolus of ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) a patient affected by immunoblastic lymphoma underwent a neurotoxic crisis. The episode lasted 1 week and was followed by a dramatic fall in plasma sodium (104 mEq I-1), associated with a proportionally lesser decrease in plasma chloride and phosphate. Despite the lowest plasma osmolality, detectable levels of circulating ADH were present. After 36 h the hyponatremic episode improved after the infusion of hypertonic sodium chloride. Nevertheless the patient lapsed into a hypotonic coma. The urinary concentrations of the main tubular enzymes (gamma-glutamyltranspeptidase, N-acetyl-glucosaminidase, alpha-glucosidase) proved very high and successively decreased slowly. The most likely cause of such hyponatremic episode is vinblastine. The drug acted through: (a) an already known inappropriate release of ADH, and (b) a hitherto unreported tubular lesion, which impaired the reabsorption of sodium and other coupled solutes.

Specificity of carcinoembryonic antigen, gastrointestinal cancer-associated antigen, tissue polypeptide antigen, fibrinopeptide A and gamma-glutamyltransferase in the diagnosis and follow-up of gastric cancer.

The usefulness and specificity of the main tumor markers (carcinoembryonic antigen, CEA; gastrointestinal cancer-associated antigen, GICA; tissue polypeptide antigen, TPA; fibrinopeptide A, FpA; gamma-glutamyltransferase, gamma-GT) have been investigated in the diagnosis and follow-up of the circumscribed and disseminated gastric cancers (GCs). The comprehensive evaluation of all of these markers has given the most reliable results. For the diagnosis and follow-up of GCs, the present study has shown that the sensitivity and specificity of the above markers have the following decreasing order: FpA, TPA, GICA, CEA, gamma-GT. However gamma-GT has proved to be a reliable index of the presence of hepatic metastases.

Tubule recovery after obstructive nephropathy relief: the value of enzymuria and microproteinuria.

The recovery of tubules after relief of obstructive nephropathy may be investigated through serial assessment of the urinary excretion of tubular enzymes alpha-glucosidase, gamma-glutamyl-transferase and N-acetyl glucosaminidase as well as of the microprotein beta-2-microglobulin. We studied 21 patients in whom obstructive nephropathy was relieved by operative or nonoperative methods. Anuria persisted from 2 to 14 days. In these patients urinary excretion of alpha-glucosidase, gamma-glutamyl-transferase, N-acetyl glucosaminidase and beta-2-microglobulin, as well as the serum creatinine were assessed weekly. Serum creatinine was the earliest index to return to normal (within 9 to 26 days). Enzymuria returned to normal within 35 to 45 days, whereas normal urinary excretion of beta-2-microglobulin occurred more than 100 days after relief of obstructive nephropathy. N-acetyl glucosaminidase and gamma-glutamyl-transferase proved to be more reliable than alpha-glucosidase in detecting recovery of the luminal membrane of the proximal tubule. The return to normal of urinary beta-2-microglobulin levels has been shown to occur later, since more specific and complex intracellular functions underlie this index. The pathophysiological aspects of recovery of obstructive nephropathy may be considered similar to those observed in ischemic acute renal failure, since in both instances hemodynamic changes are involved.

Specificity of fibrinopeptide A (FpA) as a marker for gastrointestinal cancers before and after surgery.

In 70 patients affected by gastrointestinal malignancies the plasma fibrinopeptide A (FpA) levels were assessed both before and at various intervals after the operation. At the same time other more commonly used tests of coagulation were carried out. In all the patients plasma FpA levels were shown to be variously elevated, so that they could give useful clues to the diagnosis, treatment, prognosis and follow-up of the gastrointestinal malignancies. In all the patients the coagulation tests fell into the normal ranges. However in the patients affected by recto-sigmoid adenocarcinoma an increase of circulating fibrin degradation products (FDPs) was observed which paralleled the increase of plasma FpA. In conclusion, in gastrointestinal malignancies the increase of plasma FpA levels suggests the cancer-induced start of the coagulation cascade. The assay of this peptide proves to be a reliable marker for these diseases.

Are the vascular complications of diabetes mellitus preceded by an altered thromboxane/prostacyclin plasmatic ratio?

Although many data regarding the biosynthesis of thromboxane A2 and prostacyclin in diabetes mellitus have recently appeared in the literature, it is not clear whether an imbalance between the generation of the two prostaglandins might be connected to the vascular complications of diabetes. In the present review we have tried to emphasize the most significant aspects of these studies and we have focused on alterations of platelet prostacyclin receptors and on the effects of circulating immune complexes on platelets of diabetics. It is likely that studies on the release of platelet derived growth factor as well as more precise definitions of its action on vessel wall cells leading to a massive release of prostacyclin, will permit us to ascertain whether an alteration in prostaglandin ratio is linked to the genesis of the vascular complications in diabetics.

Evidence for glycoprotein abnormality in platelets from patients with May-Hegglin anomaly.

In the present research protein analysis on SDS-polyacrylamide gel electrophoresis (PAGE) has been used to study the glycoprotein pattern of the blood platelets of four members from a family affected by May-Hegglin anomaly. In order to characterize the glycoprotein fractions, lactoperoxidase-iodination and immunoprecipitation procedures were used. N,N'diallyltartardiamide (DATD) cross-linked gel electrophoresis was shown to improve the glycoprotein pattern resolution, although with the lactoperoxidase-iodination the glycoprotein characterization of May-Hegglin platelets overlapped the normal. On the other hand with the immunoprecipitation the specific antiserum precipitated all the five major fractions of normal membrane glycoproteins, but it was shown to react quite poorly with the component V of the May-Hegglin glycoprotein pattern.

Platelet aggregation and evaluation of the ratio thromboxane B2/6-keto-prostaglandin F1 alpha in the plasma of patients on long term cimetidine treatment.

It has been reported that a long term treatment with cimetidine may give rise to thrombotic complications and may cause reversible damage to blood cells. In 57 patients on long term cimetidine treatment, platelet aggregates, platelet aggregation in vitro, plasma 6-keto-PGF1 alpha/thromboxane B2 ratio and platelet cyclic AMP levels were assessed. In 52% of the patients, platelet aggregate ratios were abnormal and collagen and ADP-hypersensitive platelets were observed. Such alterations began occurring after the first month of therapy and were shown to worsen progressively during the administration. Four of these patients, who developed unexpected thrombotic compliances after about 7 months of therapy, showed higher than normal plasma thromboxane B2, lower plasma 6-keto-PGF1 alpha and two of them, lower platelet cyclic AMP concentrations. It is suggested that cimetidine, through an unknown mechanism which probably involves activation of endogenous cyclic AMP phosphodiesterase, may favour the action of platelet aggregating agents.

Sickling and Vinblastine.

After incubation of sickle anaemia (SS) erythrocytes for 1 hour at room temperature with Vinblastine (VBL) doses (0.2 mM) far lower than those capable of causing spherostomatocytosis of normal red blood cells (0.6 mM), the percent of erythrocytes which underwent swelling resulted to be overlapping the percentage of cells which showed irreversible sickling (IS) after adequate deoxygenation. Thus VBL can reveal in the SS erythrocytes the most liable cohort of cells that show IS; this may be accounted for by the fact that in both irreversibly sickled and VBL-incubated erythrocytes there are overlapping disorders of the membrane cationic traffic. The increase of the drug concentrations induced an exponentially related cohort of SS erythrocytes to swell. The observed failure of SS erythrocytes previously incubated with VBL to sickle after adequate deoxygenation is accounted for by the close interaction of the drug with the red cell membrane subunits, which renders the red cell surface unliable to undergo further morphological alterations.

Human renin granules: ultrastructural aspects.

The ultrastructure of human renin granules has been studied from a case of Bartter's syndrome. Renin bodies present roundish (R), lobulated (L) or sharply angulated (SA) shapes. The latter are indicative of the highest rates of renin synthesis and thereby of the greatest concentrations of the pressor enzyme. Their singular shape is consistent with the form monoclinic crystals assume when they grow in an isotrophic medium. R granules form owing to filling of Golgian vesicles by renin. R bodies may then coalesce for giving rise to transient L forms. Renin is released through leak from the containing granules into the intracellular space.