Use of Glucose Control Indicators for Risk Assessment and Treatment of Neurodevelopmental Disorders and Techniques for Establishing the Status of Chronic Glucose Control

Abstract

Dysglycemia as a risk factor for neurodevelopmental disorder or developmental diabetes. The risk is assessed based on measurement of a glucose control indicator in a blood sample. One particular example of a neurodevelopmental disorder is retinopathy of prematurity in an infant. One particular example of a glucose control indicator is ‘comprehensive glycated hemoglobin fraction’ or ‘comprehensive glycated albumin fraction.’ This is calculated using ‘total whole blood protein’ in the denominator. In the case of chronic hyperglycemia, there is an increased risk of proliferative retinopathy of prematurity. In the case of chronic hypoglycemia, there is an increased risk of non-proliferative retinopathy of prematurity. This ‘total whole blood protein’ technique could also be used to determine the glucose control status in other types of patients.

Claims

1. A method for assessing the risk of a neurodevelopmental disorder in a person, comprising: having a blood sample of the person; measuring a glucose control indicator in the blood sample; comparing the measured glucose control indicator to a reference level; based on the comparison, determining the risk of developing a neurodevelopmental disorder.

2. The method of claim 1, wherein the glucose control indicator is a glycated hemoglobin fraction; wherein the step of measuring the glucose control indicator comprises assaying the blood sample for glycated hemoglobin and total protein; wherein the glycated hemoglobin fraction is calculated as the amount of glycated hemoglobin relative to total protein.

3. The method of claim 2, wherein the glucose control indicator is comprehensive glycated hemoglobin fraction; wherein the total protein is total whole blood protein.

4. The method of claim 1, wherein the glucose control indicator is glycated albumin fraction; wherein the step of measuring the glucose control indicator comprises assaying the blood sample for glycated albumin and total protein; wherein the glycated hemoglobin fraction is calculated as the amount of glycated albumin relative to the total protein.

5. The method of claim 4, wherein the glucose control indicator is comprehensive glycated albumin fraction; wherein the total protein is total whole blood protein.

6. The method of claim 1, wherein the person is a preterm infant and the neurodevelopmental disorder is retinopathy of prematurity.

7. The method of claim 6, wherein the glucose control indicator being below the reference level indicates a risk of developing non-proliferative retinopathy of prematurity.

8. The method of claim 6, wherein the glucose control indicator being above the reference level indicates a risk of developing proliferative retinopathy of prematurity.

9. The method of claim 1, wherein the person is an infant and the neurodevelopmental disorder is intraventricular hemorrhage.

10. The method of claim 1, wherein the person is a fetus and the blood sample is obtained from the mother.

11. The method of claim 1, wherein the blood sample is provided as a dried blood spot.

12. The method of claim 11, further comprising eluting the dried blood spot into a liquid fluid, wherein measurement of the glucose control indicator is performed on the eluted fluid; and further comprising measuring total protein in the eluted fluid.

13. The method of claim 1, wherein the person is an infant, and further comprising having multiple blood samples obtained from different times; wherein at least one blood sample is obtained before birth, at birth, within a week of birth, or at about one week age; wherein at least another blood sample is obtained at about four weeks age or after; wherein the glucose control indicator is measured from each one of the multiple blood samples.

14. The method of claim 1, wherein the method is further for treating the person at risk of developing a neurodevelopmental disorder, and the method further comprises: in the situation where the glucose control indicator is above the reference level, performing the step of: (i) initiating a glucose lowering medication, (ii) increasing the dosage thereof, or (iii) reducing or eliminating or modifying any glucose supplementation that the person is receiving; in the situation where the glucose control indicator is below the reference level, performing the step of: (i) initiating administration of a glucose or dextrose nourishment, or (ii) increasing the amount thereof.

15. A method of determining a glucose control status in a person, comprising: having a blood sample of the person; measuring the amount of glycated hemoglobin or glycated albumin in the blood sample; measuring the amount of total whole blood protein in the blood sample; calculating a comprehensive glycated hemoglobin fraction or a comprehensive glycated albumin fraction as the amount of glycated hemoglobin or amount of glycated albumin, respectively, relative to total whole blood protein; comparing the comprehensive glycated hemoglobin fraction or the comprehensive glycated albumin fraction to a reference level; based on the comparison, determining the chronic glucose control status of the patient.

16. The method of claim 15, wherein the glucose control indicator being above the reference level indicates a diagnosis of chronic hyperglycemia; and wherein the method is further for treating the person for chronic hyperglycemia by administering a glucose lowering medication or with exercise and diet.

17. The method of claim 15, wherein the person is a pregnant or post-partum woman and the chronic hyperglycemia is from gestational diabetes.

18. The method of claim 15, further comprising having multiple blood samples obtained from different times; wherein at least one blood sample is used to make a diagnosis of a dysglycemic medical condition or establish a baseline value for the glucose control indicator; wherein a subsequent blood sample is obtained at least four weeks later; wherein the comprehensive glycated hemoglobin fraction or the comprehensive glycated albumin fraction is calculated as results from each one of the multiple blood samples; and optionally, wherein the method further comprises (i) using the at least one blood sample to determine a treatment plan for the dysglycemic medical condition; and (ii) comparing the results to one another to monitor and adjust the treatment plan.

19. A method for assessing the risk of developmental diabetes in a fetus or infant, comprising: having a blood sample; measuring a glucose control indicator in the blood sample; comparing the measured glucose control indicator to a reference level; wherein the glucose control indicator being above the reference level indicates a risk of developing developmental diabetes

20. The method of claim 19, wherein the method is further for treating the fetus or infant at risk of developing a neurodevelopmental disorder; and the method further comprises performing the step of, on the mother or infant: (i) initiating a glucose lowering medication, (ii) increasing the dosage thereof, or (iii) reducing or eliminating any glucose supplementation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 shows the results of the preterm infant study that was performed.

DETAILED DESCRIPTION OF EXAMPLES EMBODIMENT

[0039] To assist in understanding the invention, the following example embodiments are described in more detail. Example Procedure: Collect blood from the patient (which could also be maternal, placental, or umbilical cord blood, as explained above). The blood can be either liquid blood or blood dried on filter paper (which is a dried blood spot). If the blood is dried on filter paper, elute the blood from the filter paper and perform measurements on the eluted fluid. Lyse the red blood cells in the blood sample using any suitable technique, such as immersing in lysis buffer. Measure the amount of glycated hemoglobin using any conventional technique, such as high-pressure liquid chromatography, boronate affinity, or immunoassay. Measure the amount of total protein in the cell-lysed blood sample using any conventional technique, such as by Biuret reagent, Kjeldahl method, dye-binding, or refractometry. One particular example is the “Micro BCA Protein Assay Kit” sold by various laboratory suppliers. Use appropriate statistical analysis to interpret the results. Because the results represent approximately one month (prior) of blood glucose control levels, repeat the testing at multiple different times, such as at or near birth (cord blood or within a week of birth), one week old, four weeks old, eight weeks old, 12 weeks old, etc.

[0040] Experimental Work: This was a cross-sectional study of 43 preterm infants born at 26-28 weeks gestational age. For each infant, blood samples were drawn after birth at <1 week chronological age, and again at approximately 4 weeks of age. These blood samples were stored as dried blood spots (DBS) for further analysis. In preparation for measuring glycated hemoglobin (HbA1c), the DBS were soaked in 120 μl of RBC lysis buffer for 60 min at room temperature. Samples were centrifuged at 13,000 rpm for 5 min at 4° C. Supernatants were collected and stored on ice. The resulting liquid samples were diluted 1:4000 with sample diluent (supplied with kit).

[0041] Glycated hemoglobin (HbA1c) in the liquid samples (DBS extracts) were measured by ELISA. 50 μl of standard and samples, and 50 μl of biotin-conjugate solution (except blank wells) were put in ELISA plate wells. Plates were sealed and incubated for 40 min at 37° C. Plate wells were decanted and washed with wash buffer. 100 μl of HRP-conjugate solution was then added to all the wells (except blank wells). The plates were sealed and incubated for 40 min at 37° C. The plate wells were washed with wash buffer. 90 μl of substrate solution added to each well. The plates were sealed and incubated for 20 min at 37° C. in dark. To stop the color development, 50 μl of stop solution was added to each well. The plate was immediately read with 450 nm light.

[0042] This study sought to find correlations with the relative amount of HbA1c expressed as concentration in relation to the relative amount of total whole blood protein in the DBS extract. Towards this objective, the total whole blood protein (mg) contained in the DBS extracts were measured using a BCA protein assay kit. FIG. 1 shows the results of this study. The bar graphs indicate the relative hemoglobin A1c levels in the various groups. To produce the bar graphs, the data from each assay were normalized to the mean of the matched controls (i.e. premature infants with no ROP). In other words, the controls were set to 1.0 on a relative HbA1c scale. Non-parametric matched t-tests were utilized for statistical comparisons between matched pairs. Each graph represents pairs of infants (no ROP vs non-proliferative ROP seen in top graphs; no ROP versus proliferative ROP seen in bottom graphs) matched by sex and birthweight. NS=non-significant; *p<0.05, **p<0.01, ***p<0.001.

[0043] In addition to humans, this invention could also be used in other mammalian animals as well. The descriptions and examples given herein are intended merely to illustrate the invention and are not intended to be limiting. Each of the disclosed aspects and embodiments of the invention may be considered individually or in combination with other aspects, embodiments, and variations of the invention. In addition, unless otherwise specified, the steps of the methods of the invention are not confined to any particular order of performance. Modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, and such modifications are within the scope of the invention.

[0044] Any use of the word “or” herein is intended to be inclusive and is equivalent to the expression “and/or,” unless the context clearly dictates otherwise. As such, for example, the expression “A or B” means A, or B, or both A and B. Similarly, for example, the expression “A, B, or C” means A, or B, or C, or any combination thereof.

[0045] Hypothetical Insights: I propose various insights about the relationship between blood glucose, diabetes, and ROP. However, these hypotheses are for informative and academic purposes only and are not intended to limit the invention in any way. I teach that ROP is not one disease with two phases (as currently believed). Instead, I teach that ROP, like cancer, is actually multiple diseases. I teach that a novel disorder that I will call ‘hypoglycemic ROP’ is one type of ROP disease. I teach that the cases fitting the hypoglycemic ROP disease category are those that are currently classified as mild ROP, ROP Stage 1 or Stage 2 or non-proliferative ROP (NP-ROP) which regress (and do not progress to the proliferative stages).

[0046] I teach that ‘hypoglycemic ROP’ can be a clinical marker of hypoglycemia in preterm infants to establish reference levels for chronic hypoglycemia (of about a month's duration) in preterm infants as part of a metric to estimate glucose control. It is because of the lack of any clinical cut-off points to establish criteria for hypoglycemia that the hemoglobin A1c test (and other glucose control metrics) are not currently used to monitor chronic hypoglycemia.

[0047] I teach that it is faulty methodology in glucose evaluation that has previously led to the difficulty in establishing the link between hypoglycemia and neurodevelopmental impairment (including neuroretinal/visual disability). In prior studies, a glucose control indicator (such as described in my invention) has not been used to evaluate the association between hypoglycemia and neurodevelopment. Instead, the prior studies measured the number of hypoglycemic episodes (defined by adult reference levels for hypoglycemia) or average glucose levels. But these do not give an accurate representation of chronic glucose control.

[0048] I teach that there is an unrecognized type of diabetes that I designate as ‘developmental diabetes (DD).’ (I define ‘DD’ earlier in this patent application). Like hyperglycemia, DD is characterized by blood glucose levels that are higher than normal for gestational age. I teach that, similar to other types of diabetes, DD causes a host of diabetic-related complications affecting multiple organs (e.g. kidney, eye, brain). In other words, I teach that multiple morbidities of prematurity (such as ROP, intraventricular hemorrhage (IVH), necrotizing enterocolitis, kidney disorders, etc.) should be re-defined as diabetic complications of DD. These morbidities can be prevented by treating DD.

[0049] I teach that a disorder that I designate as ‘DD-ROP’ is a type of ROP disease. In this patent application, I refer to DD-ROP by its more traditional names “proliferative ROP” or “Late Stage ROP). I teach that DD-ROP can be used as a clinical marker of DD in preterm infants to establish reference levels for chronic hyperglycemia associated with DD, in preterm infants as part of a metric to estimate glucose control. It can be used in any methodology for assessing glucose control, such as the described in this patent application or in other glucose control metrics. I teach that the cases fitting the DD-ROP disease category type are those that are currently classified as proliferative ROP. I teach that DD-ROP can be used in the methodology for assessing glucose control described in this patent application. I teach that my glycated hemoglobin measurement technique can be used to estimate the risk of short term (within the first 6 months of life) and long-term complications from DD. These complications include neuro-retinal diseases (e.g. proliferative ROP) as well as various types of neurodevelopmental impairment.

[0050] I teach that it is faulty methodology in glucose evaluation that has previously led to the difficulty in establishing the link between hyperglycemia and proliferative ROP. In previous studies, chronic glucose control indicators were not used to evaluate the association between ROP and chronic hyperglycemia. Instead, they measured the number of hyperglycemic episodes (defined by adult reference levels for hyperglycemia) or average glucose levels. Because these are surrogate measures of hyperglycemia, they do not give an accurate representation of chronic glucose control.