Results of pancreatic blood shunting into the systemic blood flow in insulin-dependent diabetics.

A new surgical method of treating patients with unstable insulin-dependent diabetes (IDD) has been developed--that of surgically shunting pancreatic blood into the systemic blood flow with the purpose of creating a more optimal interaction of subcutaneously administered insulin and pancreas-secreted glucagon. The long term results of the operation depend on the patency of a splenorenal anastomosis. This has been studied by following up 137 patients over periods from half a year to three years. Anastomotic patency was determined by renal and splenic venography and celiacy arteriography, which revealed a patent anastomosis in 114 patients, and an obliterated one in 23. Patients with patent anastomoses showed a lowering of glycosylated hemoglobin (HbA1c) from 13.3 +/- 0.3% to 9.3 +/- 0.6%, p < 0.05, a decrease of the injected insulin dose from 0.97 +/- 0.04 to 0.72 +/- 0.03 U/kg, p < 0.05, disappearance or considerable abatement of pain in the lower extremities, and of hypoglycemia. Improvement of clinical status was accompanied by an increase of glucagon in the systemic blood stream from 60.8 +/- 10.1 to 91.5 +/- 9.4 pg/ml, p < 0.05, a rise of tissue oxygen pressure, pO2, from 49.2 +/- 2.4 to 58.1 +/- 1.9 mm Hg, p < 0.05. In patients with oblivious anastomoses postoperative HbA1c levels did not change from preoperative values: 12.9 +/- 0.4% and 12.8 +/- 0.7%, p < 0.05, respectively; the insulin dose remained the same--0.91 +/- 0.07 U/kg and 0.85 +/- 0.07 U/kg, p < 0.05, no rise of the systemic blood glucagon content was noted, and former complaints continued. The suggested method is not an alternative for insulin therapy, but considerably enhances its potential.

[Urinary enzymes in insulin-dependent diabetes mellitus]. / K voprosu o fermenturii pri insulinzavisimom sakharnom diabete.

In patients suffering from insulin-dependent diabetes mellitus (IDDM) with or without preclinical and clinical signs of diabetic nephropathy, the degree of epithelial cell lesions in the renal tubules was assessed from the urinary activities of enzymes at various sites, such as lysosomal (N-acetyl-beta-D-glucosaminidase (NAG) and beta-galactosidase (beta-GA)), brush edge membranous (alanine aminopeptidase (AAP), and cytosolic (alpha-glucosidase (alpha-GL)). Patients from Groups 1 and 2 had no preclinical and clinical signs of nephropathy. In Group 1 patients, the magnitude of enzymuria was not different from that in normalcy. However, Group 2 patients exhibited significant increases in urinary NAG and beta-GA activities as compared to Group 1 patients and healthy individuals. In Group 3 patients with microproteinuria from 0.05 to 0.5 mg protein per ml urine, displayed a further enhancement of NAG and beta-GA activities as compared to Group 2 patients and significantly higher activity than did Groups 1 and 2 patients and healthy individuals. In Group 4 patients with macroproteinuria of > 0.5 mg/ml), greater increases in the activities of NAG, beta-GA, and AAP were not found, however, there was a significant increase in alpha-G1 activity. The findings suggest the varying degrees of epithelial cell damage in the renal tubules in patients of different groups and the possibility of early detection of lesion in the proximal portion of nephronic tubules in IDDM patients as assessed from urinary enzyme levels.

[Evaluation of proteinuria by ponceau S dye in patients with insulin-dependent diabetes mellitus]. / Otsenka proteinurii s ispol'zovaniem krasitelia ponso S u bol'nykh insulinzavisimym sakharnym diabetom.

Protein content was measured using ponceau S stain in "protein-free" urine (as shown by clinical analysis with sulfosalicylic acid) of 34 patients with insulin-dependent diabetes mellitus. In 15 patients (group 1) protein levels did not differ from those in healthy subjects. In 19 patients (group 2) protein levels surpassed the normal value by 3.6 times, on an average (p < 0.001). Measurements of urinary albumin revealed microalbuminuria in group 2 and normo-albuminuria in group 1. The data indicate that protein measured with the use of ponceau S stain in group 2 patients is not a false-positive result, but reflects the presence of proteins of plasma origin in the urine. Hence, measurements of urinary protein with the use of ponceau S stain may be used for identification of early stages of diabetic nephropathy.

[Two forms of aldose reductase in erythrocytes from patients with insulin-dependent diabetes mellitus]. / Dve formy al'dozoreduktazy v éritrotsitakh bol'nykh insulinzavisimym sakharnym diabetom .

Erythrocytes of patients with insulin-dependent diabetes mellitus contained two forms of aldose reductase a and b (EC No. 1.1.1.21) (the key enzyme in the sorbitol pathway of glucose utilization) as compared with those of healthy donors in whom the form b of the enzyme was only found. The aldose reductase a exhibited a higher maximal rate (Vmax = 16.7 +/- 3.2 IU/D280) and a lower substrate affinity (Km = 6.5 = 19 mM) than the enzyme b, Vmax = 43.8 +/- 0.6 IU/D280, Km = 3.0 = 4.0 mM, respectively. More severe development of the disease was observed in the patients whose erythrocytes contained only aldose reductase a (HbA1c = 14.56 +/- 0.69%) as compared with those in whom the enzyme a and b were found (HbA1c = 11.5 +/- 0.4%). Kinetic parameters of the enzyme showed that aldose reductase a may be active in hyperglycemia of diabetes mellitus, thus contributing to intensification of the sorbitol pathway in these patients.

[Determination of glycosylated hemoglobin by affinity chromatography on boron phenylagarose]. / Opredelenie glikozilirovannogo gemoglobina metodom affinnoi khromatografii na borfenilagaroze.

When Hb AIc was estimated by affinity chromatography where boron phenyl-agarose was used as a sorbent, the highest rate of Hb AIc binding with the sorbent was detected at pH 8.5-9.0 while the maximal elution of the protein occurred at 0.3-0.5 M concentration of sorbitol used as an eluent. Content of Hb AIc, estimated in 56 patients with insulin-dependent diabetes mellitus, constituted 12.3 +/- 0.4%, while in 20 healthy volunteers it was equal to 5.4 +/- 0.2%, which are consistent with the literature data obtained by other methods. At the same time, the data of Hb AIc estimation by means of affinity chromatography correlated exactly with the results of ion exchange chromatography (r = 0.98), thus corroborating the validity of the procedure used. Only the stable ketoamine fraction of Hb AIc was found to interact with boron phenyl-agarose.

[New dipeptide fluorogenic substrates of human tissue kallikrein]. / Novye dipeptidnye fluorogennye substraty tkanevogo kallikreina cheloveka.

Based on 4-methylcoumarinyl-7-amide (Amc) arginine and a series N-alkyloxycarbonyl derivatives of phenylalanine, eleven Amc-derivatives of the type ROCO-Phe-Arg-Amc (R = alkyl) were synthesized; also were n-C3H7OCO-Leu-Arg-Amc and n-C3H7OCO-D-Phe-Arg-Amc synthesized. The enzymatic hydrolysis of these compounds under the action of tissue and plasma human kallikreins were studied. Tissue kallikrein from human urine hydrolyzed the compounds with R = n-propyl and n-butyl and n-C3H7OCO-Leu-Arg-Amc more readily than the known substrates Z-Phe-Arg-Amc and H-Pro-Phe-Arg-Amc. n-C3H7OCO-D-Phe-Arg-Amc is a weak inhibitor of this enzyme (Ki = 1.5.10(-4) M). Human plasma kallikrein hydrolyzed these novel substrates at a lower rate than Z-Phe-Arg-Amc.

[Purification and some physico-chemical and enzymatic properties of tissue kallikrein from human urine]. / Ochistka i nekotorye fiziko-khimicheskie i énzimaticheskie svoistva tkanevogo kallikreina iz mochi cheloveka.

A procedure for obtaining tissue kallikrein (EC 3.4.21.35) from large specimens of human urea (100 l) has been developed. The isolation procedure included primary extraction of the protein with chitosan (a crustacean chitin deacylated by alkaline treatment), desorption from chitosan with 1 M NH3, affinity chromatography on contrical-Sepharose, ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephadex G-100. This method permits to obtain tissue kallikrein preparations purified 1080-fold (with respect to AcPheArg-OEt esterase) and 1360-fold (with respect to kininogenase) with 33 and 40% yields, respectively. Tissue kallikrein preparations were homogeneous as could be judged from the results of electrophoresis performed in 12% PAAG in the presence of 0.1% SDS as well as from the presence of one N-terminal amino acid identified as isoleucine. Purified tissue kallikrein had specific activities of 133 mumol/min/mg protein (with respect to AcPheArg-OEt hydrolysis) and 8.8 mumol/min/mg protein (with respect to D-Val-Leu-Arg-pNa hydrolysis) and liberated 462 micrograms equiv. of bradykinin/min/mg protein from heated human blood plasma used as a kininogen source. The protein exhibited the highest stability at pH 8.0-9.0; the pH optimum is at pH 8.0 with AcPheArg-OMe as substrate. The enzyme revealed a high thermostability and was fully inactivated only after 1-hour heating in a boiling water bath. The identity of the urine enzyme to tissue kallikrein could be confirmed by the resistance of the enzyme activity to SIT, high sensitivity to the inhibiting effect of aprotinin (Ki = 0.94 x 10(-10) M) and by an exceedingly low value of the second order inhibition constant for DPP (4.6 M-1 min-1). The fact that this value differs drastically from that for human blood plasma kallikrein (EC 3.4.21.34) which is equal to 360 M-1 min-1 points to marked differences in the structure of the active centers of the both kallikreins as well as to the uniqueness of the tissue kallikrein active center.

[Photometric method of determination of the amidase activity of proteinases using 4-methylcoumaryl-7-amide substrates]. / Fotometricheskii metod opredeleniia amidaznoi aktivnosti proteinaz s ispol'zovaniem 4-metilkumaril-7-amidnykh substratov.

It was shown that 7-amino-4-methylcoumarin (MC-amine), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10,800 M-1 cm-1.

Mitogenic effect of kallikrein from human urine on cultured human skin fibroblasts. Analysis of the combined action of kallikrein, insulin, and fibroblast growth factor on DNA and RNA synthesis.

Kallikrein isolated from human urine was capable of stimulating DNA and RNA synthesis in cultured human skin fibroblasts in media with a low serum content. The same concentration of kallikrein had a different effect on the DNA and RNA synthesis in different fibroblast lines, which was attributed to differences in the sensitivity of the cells to kallikrein. At a dose of 0.5 micrograms ml-1, kallikrein inactivated by heating at 100 degrees C caused an abrupt decrease in DNA synthesis in all the cell lines studied. When either active or inactivated kallikrein was added to the growth medium simultaneously with insulin there was a competitive effect on DNA and RNA synthesis. Preincubation of the cells with kallikrein prior to addition of insulin led to a reduction in the level of DNA synthesis compared to that seen upon simultaneous addition of kallikrein and insulin, suggesting that kallikrein and insulin competed for the same receptor. When kallikrein and fibroblast growth factor (FGF) were added simultaneously to the growth medium, there was a sharp decrease in both DNA and RNA synthesis in the cells compared to that seen on addition of FGF alone. Since heparin protected FGF from kallikrein inactivation, it is suggested that inactivation was caused by proteolytic degradation of part of the FGF molecule by kallikrein. It is concluded that kallikrein and insulin compete for the same receptor, possibly the insulin-like growth factor I (IGF-I), and that binding of kallikrein to this receptor is a prerequisite for mediation of the stimulatory effect of kallikrein on nucleic acid synthesis.

[Effective method of simultaneous extraction from the urine and purification of tissue kallikrein and acid-stable trypsin inhibitor]. / Effektivnyi sposob odnovremennogo izvlecheniia iz mochi i ochistki tkanevogo kallikreina i kislotostabil'nogo ingibitora tripsina.

A simple preparative procedure is developed for simultaneous isolation from urine of tissue kallikrein and acid stable trypsin inhibitor. The procedure involved adsorption of these proteins on chitosan at pH 5-6 and the subsequent elution with 1 N NH4OH, which enabled to obtain the enzyme and inhibitor with a yield of 80-90% and to purify 10-fold each of these components. Use of chitosan facilitated and simplified distinctly the large scale isolation from urine of the kallikrein and trypsin inhibitor, required for medicinal and diagnostic purposes.

[A case of hereditary angioneurotic edema provoked by functional insufficiency of C1-inactivator]. / Sluchai nasledstvennogo angionevroticheskkogo oteka, obuslovlennogo funktsional'noi nedostatochnost'iu C1- inaktivatora.

The hemolytic activity of complement, the concentration of C1-inactivator, of C1q and C4 components of complement as well as the activity of contact-activated kallikrein and the functional activity of C1-inactivator were studied in the plasma of a female patient with hereditary angioneurotic edema (HAE), of her mother, sister and normal donors. The hemolytic activity of complement, the concentration of C1-inactivator and C1q in the blood plasma of the persons examined turned out unchanged as compared with the same parameters in donors. Meanwhile in the patient with HAE and her mother with symptoms similar to HAE, the concentration of C4 appeared lower than that in the patient's sister not suffering from the disease and in donors. The activity of contact-activated kallikrein in the plasma of the patient's sister and mother exceeded the activity of the enzyme in donors. The least activity of plasma kallikrein was recorded in the proband. The functional activity of C1-inactivator in the proband constituted 50-60% of the activity of that parameter in her sister and donors. The magnitude of the functional activity of C1-inactivator in the patient's mother approximated its magnitude in the proband. Therefore, the development of HAE in the given case was mainly provoked by functional insufficiency of C1-inactivator. The magnitude of the spontaneous TAME-esterase kallikrein-like activity in the blood plasma of the patient with HAE remained within normal.

[Inhibition of kallikrein from human plasma by salivary gland secretion and extracts of the medicinal leech Hirudo medicinalis]. / Ingibirovanie kallikreina plazmy krovi cheloveka sekretom sliunnykh zhelez i ékstraktom iz meditsinskoi piiavki Hirudo medicinalis.

It has been shown for the first time that the salivary gland secretion of the medicinal leech (Hirudo medicinalis) contains a human blood plasma kallikrein inhibitor which is capable of blocking the amidolytic activity of the enzyme in an irreversible manner (with D-Pro-Phe-Arg-pNA as substrate) and which also suppresses the kininogenase activity of kallikrein. The inhibition of the amidolytic activity of highly purified kallikrein preparations from human blood plasma obeys the pseudo-first order kinetics. The experimental results suggest that in the salivary-gland secretion of H. medicinalis the inhibitor concentration exceeds by one order of magnitude that in whole leech homogenate extracts, which indicates that the inhibitor biosynthesis may be localized in leech salivary glands.

[Kinetic and allosteric properties of highly-purified, biosynthetic L-threonine dehydrogenase from brewer's yeast Saccharomyces carlsbergensis]. / Kineticheskie i allostericheskie svoistva vysokoochishchennoi "biosinteticheskoi" L-treonindegidratazy iz pivnykh drozhzhei Saccharomyces carlsbergensis.

Kinetic and allosteric propeties of highly purified "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlbergensis were studied at three pH values, using L-threonine and L-serine as substrates. It was shown that the plot of the initial reaction rate (v) versus initial substrate concentrations ([S]0 pH 6.5 is hyperbolic (Km=5.0.10-2M), while these at pH 7.8 and 9.5 have a faintly pronounced sigmoidal shape with fast occurring saturation plateaus ([S]0.5= 1.0.10-2 and 0.9.10-2M, respectively). the ratios between L-threonine and L-serine dehydratation rates depend on pH. The kinetic properties and the dependence of substrate specificity on pH suggest that the enzyme molecule undergoes pH-induced (at pH 7.0) conformational changes. The determination of pK values of the enzyme functional groups involved in L-threonine binding demonstrated that these groups have pK is approximately equal to 7.5 and 9.5. The latter group was hypothetically identified as a epsilon-NH2-group of the lysine residue. High concentrations of the allosteric inhibitor (L-isoleucine) decrease the rates of L-threonine and L-serine dehydratation and induce the appearance (at pH 6.5) or increase (at pH 7.9 and 9.5) of homotropic cooperative interactions between the active sites in the course of L-threonine dehydratation. The enzyme inhibition by L-isoleucine increases with a decrease of L-threonine concentrations. Low L-isoleucine concentrations, as well as the enzyme activator (L-valine) stimulate the enzyme at non-saturating substrate concentrations (when L-threonine or L-serine are used as substrates) without normalization of (v) versus [S]0 plots. The maximal activation of the enzyme is observed at pHG 8.5--9.0. It is assumed that the molecule of "biosynthetic" L-threonine dehydratase from brewer's yeast contains two types of sites responsible for the effector binding, i.e., "activatory" and "inhibitory" ones.

[Formation of higher alcohols by Saccharomyces carlsbergensis from branched-chain amino acids and their keto analogs]. / Obrazovanie vysshikh spirtov Saccharomyces carlsbergensis iz razvetvlennykh aminokislot i ikh ketoanalogov.

A new method of chemical synthesis of alpha-ketoisocaproic acid (the keto analogue of L-leucine) is described. It has been shown that the resting cells of Saccharomyces carlsbergensis 776, in the stationary state of biomass, produce mainly higher alcohols: isobutanol from L-valine and its keto analogue; optically active amylol only from L-isoleucine and its keto analogue; isoamylol only from L-leucine and its keto analogue. "Nonspecific" formation of n-propanol from L-valine, L-isoleucine and their keto analogues, as well as that of isobutanol from L-isoleucine and its keto analogue, has been also found at pH 7.0. Formation of higher alcohols from alpha-keto acids has an acidic pH optimum while that from L-amino acids has a neutral or a weakly alkaline pH optimum. Formation of isobutanol from L-valine is an exception. The dependence of higher alcohol formation on the pH and the kinetics of their accumulation suggest that higher alcohols are produced from L-amino acids in at least three sequential reactions: transamination, decarboxylation of the keto analogue being formed, and reduction of the aldehyde; formation of higher alcohols from alpha-keto acids involves two reactions: decarboxylation and reduction. Transamination and decarboxylation are limiting steps in the process in the former case, and decarboxylation in the latter.

[Effect of higher alcohols on the activity of biosynthetic L-threonine dehydratase from Saccharomyces carlsbergensis brewer's yeast]. / Vliianie vysshikh spirtov na aktivnost' biosinteticheskoi L-treonindegidratazy iz pivnykh drozhzhei Saccharomyces carlsbergensis.

The effects of low (1 . 10(-4) M) and high (1 . 10(-3) M) concentrations of n-propanol, isobutanol and isoamylols on the kinetic behaviour of "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlsbergensis 776 were studied. It was concluded that these alcohols control the activity of the first enzyme of the L-threonine biosynthetic pathway.