discussion of papers presented by drs. bunn, cerami, ditzel, and rubenstein

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Discussion of Papers Presented by Drs. Bunn, Cerami, Ditzel, and Rubenstein Moderator: Michael G. Page Dr. Porte: In the steady state, the data indi- cates a good relationship between HbA,, and glycemia. Has a similar correlation been estab- lished for fluctuating glycemic states? If the hypothesis is unsound, do we not risk overtreat- ment in subjects with high HbA,, levels whom the physician feels are not well controlled? Do we know that two individuals with the same HbA,, level have had the same mean glucoses? Could not one of the individuals have had very high glucoses for short periods of time and another one moderately elevated glucoses for a longer period of time? As far as methodology is concerned, the column method has to be done on reasonably fresh red cells; once the hemolysate is made, it is variably unstable. Therefore, shipment of sam- ples between laboratories is not practical, rendering interlaboratory comparisons difficult. We also had a lot of trouble with the calorimetric test, but it can be made to work. The procedure is protein sensitive, and therefore the samples have to be set up with the protein concentration adjusted for each sample. We find a very good correlation between the column and the colori- metric method under these conditions. Since the sugar in the urine is really related to overflow glycosuria, I would not expect good correlation between glycosuria and HbA,,, since HbA,, is presumably relating to plasma glucose levels. Dr. Rubenstein: Approximately 15% of our patients have a poor correlation between their HbA, and blood sugar values. Two have hemo- globin abnormalities (rapid turnover of red cells) but the others do not. Because their HbA, is not closely correlated to their blood sugar, one could make a serious mistake if significant changes in insulin therapy were made on that basis alone. With respect to methodology, thorough wash- ing and storage at -65OC has led to reproduci- ble findings. Dr. Gabbay: A decrease of 5°C in room temperature is enough to make the resin more acid and bind the HbA, more tightly. For this reason, we used the Fluckiger-Winterhalter (thiobarbituric acid) test. We find excellent Metabolism, Vol. 28, No. 4, Suppl. 1 (April), 1979 correlation (r = 0.89) between the TBA test and total HbA,,,. I anticipate that within the next six months there is going to be an even simpler calorimetric technique that directly determines the ketoamine and the glycolysine ketoamine groupings. Dr. Porte: These two tests really don’t measure the same thing, and we tend to take them under conditions that we hope will give us the same answer. Have you observed any condi- tions under which you get disagreement between the two tests? Dr. Gabball: We have a case of a 14-yr-old girl who had a 7% HbA,, level by the column technique, yet was very high by the calorimetric TBA test. She had HbC trait, and HbC chromatographs differently on the column, so her HbC,, was not measurable by the chromato- graphic technique, but was measured by the chemical method. Sickle cell hemoglobin could also be expected to show similar differences. I think that the glycosylated hemoglobin test gives a more consistent and longer-term time frame for determining what the glucose levels are doing. I think it is going to be very useful for prospective studies, but its usefulness in clinical management of the patient has yet to be explored. Dr. Cerami: I think it is possible to ship samples, if they are packed on dry ice. With the chromatographic method, oxidation of HbA will form products that will chromatograph in the HbA, peak, giving an abnormally high value. Hemoglobin F also chromatographs with HbA,, which could also give you spurious numbers. Dr. Gabbay: The calorimetric assay is hemoglobin protein concentration dependent; we use between 9 and 11 mg hemoglobin per test with excellent results. Dr. Porte: We adjust each sample individu- ally, and also run the assay at about 10 mg. Dr. Soeldner: There is no standard method for determining HbA,,, and Dr. Dayton Miller of the CDC in Atlanta is in charge of a compara- tive study of all hemoglobin assay methods, trying to determine the differences and similari- ties between them, as well as interfering drugs. 453

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Discussion of Papers Presented by Drs. Bunn, Cerami, Ditzel, and Rubenstein

Moderator: Michael G. Page

Dr. Porte: In the steady state, the data indi- cates a good relationship between HbA,, and glycemia. Has a similar correlation been estab- lished for fluctuating glycemic states? If the hypothesis is unsound, do we not risk overtreat- ment in subjects with high HbA,, levels whom the physician feels are not well controlled? Do we know that two individuals with the same HbA,, level have had the same mean glucoses? Could not one of the individuals have had very high glucoses for short periods of time and another one moderately elevated glucoses for a longer period of time?

As far as methodology is concerned, the column method has to be done on reasonably fresh red cells; once the hemolysate is made, it is variably unstable. Therefore, shipment of sam- ples between laboratories is not practical, rendering interlaboratory comparisons difficult. We also had a lot of trouble with the calorimetric test, but it can be made to work. The procedure is protein sensitive, and therefore the samples have to be set up with the protein concentration adjusted for each sample. We find a very good correlation between the column and the colori- metric method under these conditions.

Since the sugar in the urine is really related to overflow glycosuria, I would not expect good correlation between glycosuria and HbA,,, since HbA,, is presumably relating to plasma glucose levels.

Dr. Rubenstein: Approximately 15% of our patients have a poor correlation between their HbA, and blood sugar values. Two have hemo- globin abnormalities (rapid turnover of red cells) but the others do not. Because their HbA, is not closely correlated to their blood sugar, one could make a serious mistake if significant changes in insulin therapy were made on that basis alone.

With respect to methodology, thorough wash- ing and storage at -65OC has led to reproduci- ble findings.

Dr. Gabbay: A decrease of 5°C in room temperature is enough to make the resin more acid and bind the HbA, more tightly. For this reason, we used the Fluckiger-Winterhalter (thiobarbituric acid) test. We find excellent

Metabolism, Vol. 28, No. 4, Suppl. 1 (April), 1979

correlation (r = 0.89) between the TBA test and total HbA,,,. I anticipate that within the next six months there is going to be an even simpler calorimetric technique that directly determines the ketoamine and the glycolysine ketoamine groupings.

Dr. Porte: These two tests really don’t measure the same thing, and we tend to take them under conditions that we hope will give us the same answer. Have you observed any condi- tions under which you get disagreement between the two tests?

Dr. Gabball: We have a case of a 14-yr-old girl who had a 7% HbA,, level by the column technique, yet was very high by the calorimetric TBA test. She had HbC trait, and HbC chromatographs differently on the column, so her HbC,, was not measurable by the chromato- graphic technique, but was measured by the chemical method. Sickle cell hemoglobin could also be expected to show similar differences.

I think that the glycosylated hemoglobin test gives a more consistent and longer-term time frame for determining what the glucose levels are doing. I think it is going to be very useful for prospective studies, but its usefulness in clinical management of the patient has yet to be explored.

Dr. Cerami: I think it is possible to ship samples, if they are packed on dry ice. With the chromatographic method, oxidation of HbA will form products that will chromatograph in the HbA, peak, giving an abnormally high value. Hemoglobin F also chromatographs with HbA,, which could also give you spurious numbers.

Dr. Gabbay: The calorimetric assay is hemoglobin protein concentration dependent; we use between 9 and 11 mg hemoglobin per test with excellent results.

Dr. Porte: We adjust each sample individu- ally, and also run the assay at about 10 mg.

Dr. Soeldner: There is no standard method for determining HbA,,, and Dr. Dayton Miller of the CDC in Atlanta is in charge of a compara- tive study of all hemoglobin assay methods, trying to determine the differences and similari- ties between them, as well as interfering drugs.

453

454 DISCUSSION

For example, high dose aspirin ingestion, acety- lating hemoglobin, would produce something that would chromatograph rapidly, and give spurious results.

Dr. Bern: Should we expect that glycosyla- tion is more of a general problem, and should studies on glycosylation be extended to fibrino- gen, cr2-macro-globulins, and other proteins that could become dysfunctional under the influence of an attached glucose moiety? Are there any negative studies that would enhance the answer?

Dr. Cerami: Glycosylation of fibrinogen does not appear to affect its biologic activity. Overall, the amount of glycosylation is a func- tion of the turnover of the proteins. Proteins that are made to last for long periods of time are going to accumulate these glycogroups. The same comment applies to carbamylated proteins, which form from blood urea.

Dr. S. Bessman: In relation to Dr. Ditzel’s work, the K, for oxygen is so low that you could bring the tissue almost down to 1% of ordinary oxygen tension of room air and it will still oxidize with a normal rate.

Dr. Ditzel: We know a lot of things about the retina with respect to oxygen, and I think my talk was more about the retina than anything else. The retina has no sympathetic nerve system; its blood flow is only controlled by pOZ, pCO,, and pH. You can take a person and let him breathe just a tiny bit less oxygen than in the atmospheric surroundings, and immediately you will see the reaction in the small blood vessels in the retina. They dilate to a lowering of oxygen. They constrict if they have an increased oxygen in the breathing.

Dr. Fajans: Dr. Ditzel showed us that only 37% of the chemical diabetics without fasting hyperglycemia had HbA,, levels above the high- est of the normal subjects. In chemical diabetics, postprandial hyperglycemia is often not of the magnitude of the hyperglycemia following glucose ingestion during the GTT. Therefore, one should not be surprised that two thirds of these chemical diabetics have normal HbA,, levels.

Dr. Gabbay: In cyanate-induced neuropathy in rats, is there segmental demyelination?

Dr. Cahill: Yes, after 18 mo. The physio- logic nerve lesion occurs very early, and later on the morphological lesion is observed.

Dr. Cerami: At the end of 18 mo, there was a decrease in the number of nerves that had myelin. Morphological changes were apparent at 12-15 mo, when enough carbamylation had occurred on myelin proteins to be noted by electron microscopy.

Dr. Gabbay: In the diabetic rat, we don’t see segmental demyelination for up to 2 yr of diabetes. Therefore, 1 am not certain that we are dealing with the same process in the cyanate neuropathy.

Dr. Cahill: It took a dozen or so years to characterize HbA,, as being a glycosylated hemoglobin because of the chemical lability of the carbohydrate moiety. Could not this lability of glucose covalently linked as the Schiff base (which then easily dissociates) indicate a poten- tial physiologic abnormality in many other proteins around the body that could not be chemically determined after tissue isolation? Could not glucose be altering many reactions throughout the body without it being necessary to “hang on,” and go on to the irreversible Amadori rearrangement?

Dr. Gabbay: Increased glycosylation has been noted with hemoglobin, crystallin, and albumin. Once the ketoamine is formed, it is very stable, with little reversibility to the Schiff base. There are proteins that glucose will inter- act with to form a Schiff base, and then undergo an Amadori arrangement extremely rapidly; so rapidly, it has to be an enzyme, and aldose reductase turns out to be doing exactly that. The glucose is interacting with it to form a Schiff base, undergoing an Amadori rearrangement, and we can tell this by using glucose labelled with tritium on the second carbon atom.

Dr. Lebovitz: Does anyone have any data as to whether the HbA,, levels are proportionately low in hypoglycemic states, and is there a good correlation between the normal range of the blood sugar level with the HbAI,?

Dr. Rubenstein: In normal subjects, with plasma glucose values between 65 and 95 mg/dl, there is a remarkably close relationship with HbA,.

Dr. Gabbay: We’ve studied hypoglycemics, and they are not below normal. They may be down around 6%, but we don’t get much lower than that.

Dr. Lebovitz: If there is this correlation in a normal range, it is possible then that the data

DISCUSSION 455

that we saw in the chemical diabetic patients, with 37% being above the normal range, really reflects a difference in the distribution of fasting

blood sugars between that group of patients and

the controls, rather than saying that the patient with chemical diabetes really is like the diabetic.

Dr. Ditzel: In our study of 65 patients, there was a significant correlation between the area

under the glucose curve and the height of hemo- globin A,,, whereas HbA,, did not correlate with

the fasting blood glucose. In normal pregnancy cases, we have also found a nice correlation

between HbA,, and the postprandial blood

sugar, but not with the fasting blood sugar. This may suggest that the postprandial values may be

a better expression of the glucose impact on

everyday life as expressed by HbA,,. Dr. Soeldner: Two patients with docu-

mented islet cell tumors, referred to us for a brief

workup for confirmation and then removal of

their tumors, had the lowest HbA,, that we have

ever seen. Our lower limit is 4.0, the upper limit

being 6.0 in our laboratory. One patient was 3.7, the other one 3.8, lower than any other value we

have seen.