Enzymatic assay of myo-inositol in serum.

An enzymatic spectrophotometric method for determination of myo-inositol in serum is described. The method is sensitive and rapid to perform without special equipment. Linearity between the amount of myo-inositol and absorbance was obtained in the range of 0.5 to 3 nmol myo-inositol per assay. The amounts of myo-inositol determined in sera from apparently healthy subjects agree well with gas chromatographic data.

The nature of neuroendocrine abnormalities in depression: a controversial issue in contemporary psychiatry.

Neuroendocrine abnormalities in depression have been regarded, by many authors, as relatively specific markers of nosological subtypes of the disorder, e.g. primary vs. secondary, endogenous vs. non-endogenous or unipolar vs. bipolar depression. They should reflect the same changes in central neurotransmitters (e.g. noradrenergic insufficiency and/or cholinergic hyperactivity) that were hypothesized as the cause of clinical symptoms. This view is challenged on the basis of our own neuroendocrine investigations in 317 psychiatric patients and 103 normal controls. According to these studies the abnormalities are nosologically rather unspecific. They are induced by a large variety of factors, e.g. emotional stress associated with the clinical symptomatology, weight loss due to malnutrition as a consequence of reduced appetite, medication and drug withdrawal. Stress-induced hypercortisolism appears to be the most common abnormality that may trigger other neuroendocrine dysfunctions, such as a blunted TSH response to TRH. Differences in neuroendocrine abnormalities of depressives are probably due to variations in the manifold factors influencing the hormonal axes involved, to temporal changes in hormonal patterns (e.g. one abnormality triggering another) and to individual differences in the basic activity and the responsiveness of the various axes.

Nonenzymatic glycation of immunoglobulins leads to an impairment of immunoreactivity.

Incubation of purified human and rabbit immunoglobulin G with glucose leads to covalent incorporation of the sugar into the protein, depending on glucose concentration, incubation time and pH. Furthermore, the level of glycated immunoglobulin G from normal and diabetic subjects has been determined using the thiobarbituric acid reaction. The median for glycated immunoglobulin G, expressed as mmol 5-hydroxymethylfurfural per mol IgG, obtained from 20 normal and 29 diabetic subjects was 62 and 107, respectively. Glucose incubation of immunoglobulin G purified from rabbit anti-human-transferrin serum, from human anti-varicella/zoster virus serum and from human anti-lues-spirochete serum, respectively, leads to a marked decrease in biological activity, as determined in a micro complement fixation test. Inactivation of specific antibody was dependent on incubation time and glucose concentration employed. Loss in complement-fixing activity was observed at glycation levels well comparable to those found in diabetics.

Clinical utility of nonenzymatically glycosylated blood proteins as an index of glucose control.

This study compares the utility of nonenzymatically glycosylated serum proteins (lys-GSP) to glycosylated hemoglobins (HbA1a-c) as control indices of glucose homeostasis in patients with IDDM. The diagnostic value of lys-GSP was also examined in patients with non-insulin-dependent diabetes mellitus, in subjects with impaired glucose tolerance, and in two patients with insulinoma. The intraindividual fluctuation of lys-GSP in normoglycemic subjects is very small, resulting in an interindividual range of 3.0 +/- 0.3 lysine-bound glucose/mg protein (means +/- SD, N = 52). HbA1a-c with a normal range of 6.4 +/- 0.9% (N = 52) shows greater variability. In IDDM there is no overlap of lys-GSP levels between the normal and the diabetic range at the 95% confidence level. In patients treated with an open-loop insulin delivery system failure of normalization of the glucose balance was clearly discernible by an elevation of GSP. In contrast, in about 40% of the patients with incomplete glycemic control the HbA1a-c levels fell within the normal range. The utility of lys-GSP for diagnosis of diabetes is compared with the results of 60 oral glucose tolerance tests. Two patients suffering from insulinoma displayed decreased lys-GSP values. From these results it appears that determination of lys-GSP represents a more sensitive parameter for long-term control than HbA1a-c and is suitable for monitoring even small fluctuations of blood glucose.

Inactivation of bovine kidney beta-N-acetyl-D-glucosaminidase by nonenzymatic glucosylation.

Evidence is presented that the incubation of beta-N-acetyl-D-glucosaminidase from bovine kidney with glucose leads to the covalent incorporation of the sugar into the enzyme protein. Concomitantly, the enzyme activity becomes markedly reduced depending on the time of incubation and the concentration of glucose employed. The separate investigation of the isoenzymes A and B, as obtained after DEAE-cellulose chromatography of the preparation, revealed that isoenzyme A had lost some 80% of its initial activity after 15 days at 37 degrees C at 44.4mM glucose, whereas isoenzyme B activity remained unchanged. The inactivation of A was associated (1) with a marked decrease in protein stain intensity after polyacrylamide gel and Cellogel electrophoresis and (2) with the formation of a small amount of apparent isoenzyme B, as indicated by the following observations: (a) appearance of enzyme activity at the position of isoenzyme B after DEAE-cellulose chromatography, (b) appearance of protein with enzyme activity in the region of authentic isoenzyme B after Cellogel electrophoresis, (c) increase in molecular mass from 130 kDa to 205 kDa, (d) similarity in heat-stability and Km for p-nitrophenyl N-acetyl-beta-glucosaminide between glucose-treated isoenzyme A and authentic isoenzyme B. As in diabetes mellitus the activity of beta-N-acetyl-D-glucosaminidase has been reported to be decreased in kidney and other tissues, the possibility that nonenzymatic glucosylation of the enzyme might occur in vivo is considered.

Different behaviour of haemoglobin A1a-c and glycosyl-albumin levels during recovery from diabetic ketoacidosis and non-acidotic coma.

Glycosylated haemoglobin (HbA1a-c) and serum albumin (glycosyl-albumin) have been determined in patients with severe diabetic ketoacidosis and non-acidotic coma. Within one week of therapy the level of glycosyl-albumin decreased from 184 mmol 5-hydroxymethylfurfural (HMF)/mol albumin to 152 mmol HMF/mol albumin (p less than 0.01) and was gradually lowered by some 40% during a period of 17 days. In contrast, the level of HbA1a-c remained unchanged. From these observations and findings in a patient with insulinoma, it appears that glycosyl-albumin provides a more acute measure of variation in relative glycaemia than HbA1a-c, and may prove useful as a measure of medium-term diabetes control.

Improvement of the thiobarbituric acid assay for serum glycosylprotein determination.

This study shows that the thiobarbituric acid (TBA) reaction as applied to the measurement of non-enzymatically glycosylated serum proteins(s) [1], yields erroneous results unless strictly standardized conditions are followed. Critical points are in particular (a) the concentrations of NaBH4 required for the preparation of the blanks by reduction of the ketoamine linkages; (b) the amounts of protein which should be identical in all serum samples to be compared; (c) dialysis for removal of glucose and NaBH4 prior to hydrolysis; (d) increase in the yield of 5-hydroxymethylfurfural (HMF) by appropriate conditions of hydrolysis. From our data obtained with an improved TBA-assay it appears that about 90% of total glycosylated serum protein is accounted for by glycosylated albumin. Thus changes in the latter used for diagnostic purposes in diabetes, may well be reflected by total serum glycosylprotein measurement.

Increased glycosylation of serum albumin in diabetes mellitus.

The level of glycosylated albumin has been determined in the serum of normal and diabetic subjects after purification of the albumin to apparent homogeneity. The sugar was released from the albumin preparations as 5-hydroxymethylfurfural (HMF) after 24 h of hydrolysis in 2 N acetic acid at 92 degrees C, and it was assayed by the thiobarbituric acid reaction. The mean value for glycosylated albumin, expressed as picomoles of HMF per nanomole of albumin, obtained from 10 normal control and 65 diabetic subjects, was 64 and 124, respectively. The level of glycosylated albumin correlates with the mean blood glucose concentration (n = 55, r = 0.715), but not with the fasting blood sugar concentrations. Moreover, a linear relationship was observed between the amounts of glycosylated hemoglobin (HbAIa-c) and glycosyl-albumin (n = 74, r = 0.88). In an insulin-treated diabetic patient, there was a different temporal relationship between blood glucose concentrations and glycosylated hemoglobin and albumin levels. While HbAIa-c was lowered by only 15% after 20 days, glycosylated albumin had dropped by more than 50% during the same time. Our results indicate that glycosylated albumin might provide a valuable tool to assess the average blood sugar levels between shorter intervals, since the turnover of serum albumin is considerably faster than that of HbAIa-c.

[Experience with the glucose-dehydrogenase-UV-method for the determination of blood glucose (author's transl)]. / Erfahrungen mit der Glucose-Dehydrogenase-UV-Methode zur Bestimmung der Blutglucose

A method for glucose determination using glucose-dehydrogenase is described. Our experience with this method is based on 70 000 determinations of blood glucose within six months. Linearity over a wide range, accuracy and specificity highly recommend this method. No interference was noticed by substances physiologically occuring in blood. The glucose-dehydrogenase-method correlated well with the hexokinase glucose-6-phosphatedehydrogenase method for the determination of blood glucose.