Diurnal variability of glucose tetrasaccharide (Glc4) excretion in patients with glycogen storage disease type III

Abstract Aim The urinary glucose tetrasaccharide, Glcα1‐6Glcα1‐4Glcα1‐4Glc (Glc4), is a glycogen limit dextrin that is elevated in patients with glycogen storage disease (GSD) type III. We evaluated the potential of uncooked cornstarch therapy to interfere with Glc4 monitoring, by measuring the diurnal variability of Glc4 excretion in patients with GSD III. Methods Voids were collected at home over 24 hours, stored at 4°C and frozen within 48 hours. Glc4 was analyzed using liquid chromatography‐tandem mass spectrometry and normalized to creatinine. Results Subjects with GSD III (median age: 13.5 years, range: 3.7‐62; n = 18) completed one or more 24‐hour urine collection, and 28/36 collections were accepted for analysis. Glc4 was elevated in 16/18 subjects (median: 13 mmol/mol creatinine, range: 2‐75, reference range: <3). In collections with elevated Glc4 (23/28), two‐thirds (15/23) had low diurnal variability in Glc4 excretion (coefficient of variation [CV%] <25). The diurnal variability was significantly correlated with the Glc4 concentration (Pearson R = .644, P < .05), but not with the dose of uncooked cornstarch. High intraday variability (>25%) was not consistently observed in repeat collections by the same subject. Conclusions The extent and variability of Glc4 excretion relative to creatinine was not correlated with cornstarch dose. A majority of collections showed low variability over 24 hours. These findings support the use of single time‐point collections to evaluate Glc4 in patients with GSD III treated with cornstarch. However, repeat sampling over short time‐periods will provide the most accurate assessment of Glc4 excretion, as intraday variability may be increased in patients with high Glc4 excretion.


| INTRODUCTION
Glycogen storage disease type III (GSDIII, MIM #232400) is an autosomal recessive disorder caused by a deficiency of glycogen debranching enzyme (GDE, EC 3.2.1.33, EC 2.4.1.25) encoded by AGL (MIM #610860). GDE is a cytosolic enzyme that works in combination with glycogen phosphorylase to release glucose from glycogen for energy metabolism. GDE deficiency disrupts glucose homeostasis, and results in an accumulation of abnormally structured glycogen enriched in α-1-6 branch points. 1 The clinical manifestations are variable and patients are classified as GSD IIIa, characterized by liver, heart, and muscle involvement, or GSD IIIb in which the liver is predominantly affected. 2 GSD III often presents in infancy or childhood with hepatomegaly and hypoglycemia due to liver disease. Ketosis, hyperlipidemia, and growth retardation are common. 3 Skeletal muscle weakness slowly progresses in GSD IIIa, becoming more prominent in the third to fourth decades of life. Liver disease becomes less apparent in adolescence and adulthood, and is associated with a decrease in liver size and serum aminotransferase levels. 4 However, long-term hepatic complications have been reported including liver fibrosis, cirrhosis, adenomas, and hepatocellular carcinoma. 5,6 In addition to the general management of disease manifestations, treatment includes dietary therapy using uncooked cornstarch to minimize hypoglycemic events, and a high protein diet as an alternative energy source. 7,8 As new therapies are being investigated, [8][9][10] biomarkers are needed to determine the clinical severity and monitor disease progression. The glucose tetrasaccharide, Glcα1-6Glcα1-4Glcα1-4Glc (Glc 4 ) is a glycogen limit dextrin produced by circulatory amylases and neutral α1-4-glycosidases. 11 It is elevated in conditions associated with increased glycogen accumulation and/or release of glycogen from damaged tissues. [12][13][14][15][16] Urinary Glc 4 is an established biomarker in patients with Pompe disease, correlating with skeletal muscle glycogen and disease status in these patients. [17][18][19] Glc 4 is also elevated in GSD III, 16,[20][21][22][23] and has potential as a biomarker in this disorder. Glc 4 is usually measured in randomly collected voids (spot urines) for the convenience of patients and clinical personnel. However, it has not been determined whether Glc 4 measurements in spot urines represent excretion over a 24-hour period. Previous studies have suggested that ingestion of starch or glycogen may increase Glc 4 excretion, 14 and in GSD III this could be a particular concern because of cornstarch therapy. We evaluated the diurnal variability in Glc 4 excretion, to investigate whether the degree of elevation and variability in Glc 4 excretion correlated with cornstarch therapy in patients with GSD III.

| Subjects
This was a single center, prospective study of patients consented to a natural history study, approved by Duke University Health System Institutional Review Board (#Pro00047556). Patients had a confirmed diagnosis of GSD III, via AGL variant and/or enzyme analysis (Supplemental Table 1).

| Glucose tetrasaccharide and creatinine analyses
Glc 4 was analyzed as a butyl-4-aminobenzoate derivative using [ 13 C 6 ]Glc 4 as an IS, and ultraperformance liquid chromatograph-tandem mass spectrometry (UPLC-MS/ MS), as reported with modifications. 24 Urine (20 μL) was combined with 20 μL 50 μmol/L IS, incubated at 80 C for 1 hour with 152 mmol/L butyl-4-aminobenzoate, 400 mmol/L sodium cyanoborohydride, and 5.3% glacial acetic acid (vol/vol) in methanol, and excess reagent was removed using solid phase extraction. Samples were dried under nitrogen, reconstituted in 10 mmol/L ammonium acetate in 90:10 (vol:vol) acetonitrile: deionized water (diH 2 O), and separated with gradient elution on a UPLC BEH amide column using 10 mmol/L ammonium acetate in acetonitrile:diH 2 O as the mobile phase. Glc 4 and the IS were detected by selected reaction monitoring

Synopsis
The diurnal variability of Glc 4 excretion is low for a majority of patients with glycogen storage disease III and is not correlated with cornstarch dose.

| 24-hour urine collections
Urine collections over 24 hours were conducted unsupervised in a residential setting. Subjects were instructed to discard the first morning void on day 1, and collect all subsequent voids separately over the next 24 hours, ending with the first morning void on the second day. Subjects were asked to reserve a small aliquot (about 1 mL) from each void in a separate container and combine the remaining urine in a single large container. Samples were stored in a cooler on cold packs and frozen within 48 hours of collection. Glc 4 and creatinine were analyzed in each aliquot and pooled collections.

| Statistical analyses
Descriptive statistics, Pearson correlation coefficient, linear regression, paired t test, and Bland-Altman analyses were calculated using Microsoft Excel and GraphPad Prism V8. The diurnal variability of Glc 4 excretion was calculated as the CV%. P-values ≤.05 were considered significant.

| Cohort description
Here, 18 subjects (n = 2 males, subjects 13 and 15) with GSD IIIa (n = 16) or b (n = 2, subjects 17 and 20) participated in the study. The median age was 1 year (range: 0.3-12) at the time of diagnosis, and 13.5 years (range: 3.7-62) at the start of the study. All were treated with various dietary regimens of cornstarch (Table 1), except the three adults (subjects #8, 13, and 29). The protein intake goal was 20% to 25% total energy consumed for all patients with GSD IIIa, achieved in seven subjects (#2, #5, #8, #10, #12, #13, and #17) using a protein supplement, and in the remaining subjects using natural sources of protein. Most subjects were ambulatory. One adult (#29) required a wheelchair and an 8-year-old female (#27) was considering a wheelchair for long distances. year-old male (#15) required ankle-foot orthoses, an 8-year-old female (#7) was recommended to wear custom shoe inserts for calcaneal valgus, and one adult (#13) used assistive devices. Ten subjects had evidence of hepatomegaly on liver imaging. Of the three adults in the cohort, subject #8 showed evidence of liver fibrosis and cirrhosis, subject #13 underwent a multiorgan transplant (heart, liver, and kidney) after suffering heart failure, 25 and subject #29 had HCC treated by radioembolization. The liver disease natural history in the pediatric subjects was reported in detail elsewhere. 20

| 24-hour urine collections
Thirty-six 24-hour urine collections were completed by 18 subjects. Six subjects completed two and another six completed three repeat collections over 1 to 3 years. Then, 28 collections by 15 subjects were accepted for diurnal variability assessments ( Table 1) and 8 collections (22%) were excluded due to incomplete or inaccurate collection or recorded information, or inappropriate storage. The total urine volume was positively correlated with age (Pearson R: .758, P < .05) and weight (Pearson R: .700, P < .05; Supplemental Figure 1). The median number of voids was 6 (range: 4-10), the median total volume of urine collected was 1265 mL (range: 262-4000), and the median urinary output was 1.0 mL/kg/h (range: 0.53-2.7).

| Cornstarch dose and Glc 4 excretion
Glc 4 was elevated in all subjects except an 11 year old subject with GSD IIIa (#15) and a 13 year subject old with GSD IIIb (#20) ( Table 1). Glc 4 concentrations normalized to creatinine in 24-hour collections varied widely (median: 15 mmol/mol CN, range: 2-60, n = 18; calculated using median values for subjects with more than one collection) and were not significantly correlated with the total cornstarch dose ( Figure 1). Of note, both subjects with normal Glc 4 (#15 and 20) were treated with cornstarch, whereas all three subjects (#8, 13, and 29) who were not on cornstarch therapy had elevated Glc 4 ( Table 1). For the 15 subjects with acceptable 24-hour urine collections, the total amount of Glc 4 excreted in 24 hours was significantly correlated with weight, but not age (Supplemental Figure 2). In comparison, the total amount of creatinine excreted over 24 hours significantly increased with weight and age (Supplemental Figure 2).

| Variability in Glc 4 excretion over 24 hours in patients with elevated Glc 4
Glc 4 was elevated in 23/28 of the 24-hour urine collections, of which 15/23 had low (CV% ≤25), and 8/23 had higher (CV%: 26-47) diurnal variability in Glc 4 excretion (Table 1). Only 1/5 subjects who performed repeat collections (#14) had high variability in more than one collection. The diurnal variability was significantly correlated with the pooled 24-hour urine Glc 4 concentration normalized to creatinine (Pearson R = .644, P < .05), but not with the total dose of uncooked cornstarch (Figure 1). Furthermore, high variability was observed in one collection (#13-2) from a subject not on cornstarch therapy. In contrast, low variability was observed in 11 collections by 8 subjects on cornstarch therapy.
No consistent trend in Glc 4 excretion over 24 hours was observed in collections with high variability (Supplemental Figure 3). A closer agreement was observed between Glc 4 concentrations in 24-hour urines and the first void, compared with the last void collected (Supplemental Figure 4). However, the first and last voids did not differ significantly (paired t test, P = .41). For urines with low variability, both the first and last voids compared well with the 24-hour urine (Supplemental Figure 5).

| DISCUSSION
Glc 4 is a promising biomarker in GSD III as it is correlated with serum transaminases in pediatric patients with GSD III 20,23 and a GSD III dog model, 26 and with CK in adults with GSD III. 23 These observations suggest urinary Glc 4 reflects glycogen accumulation in liver more than muscle in the pediatric population, and muscle glycogen accumulation in adults. However, given the clinical variability of GSD IIIa and an increasing understanding of early muscle involvement, clinical correlation is needed in interpreting the source of Glc 4 in GSD III. Patients with GSD III are treated with uncooked cornstarch which contains amylopectin, an α1-6 branched glucose polymer that can be converted to Glc 4 by amylase activity. 27 This raises a concern for the reliability of using spot urines to assess Glc 4 excretion in cornstarch-treated patients. Our results suggest that uncooked cornstarch intake does not contribute significantly to Glc 4 excretion. A previous study investigating the impact of dietary factors on Glc 4 reported diurnal variation in the rate of excretion in a volunteer on a normal diet over three days. 14 There was evidence that the rate increased after meals; however, the overall daily excretion varied less than 10%. Decreased calorific intake and a low carbohydrate diet appeared to decrease the excretion rate, whereas a high carbohydrate diet consisting entirely of cooked rice (2400-3200 kcal/24 h), resulted in a fourfold to fivefold rate increase, compared with a regular diet. 14 The authors suggested this increase might be caused by amylopectin degradation by amylase in the gastrointestinal tract. Urinary output varies over the course of the day and is impacted by fluid intake, physical activity, and sleep. 28 Thus, an increase in the Glc 4 excretion rate under normal dietary conditions could be secondary, in part, to increased urinary output. The dose used to treat pediatric patients with GSD III is relatively low (typically 1 g/kg every 4 hours or longer, adjusted based on the ability of a dose to maintain euglycemia between feeds), 3 and many adults with GSD III have a minimal intake of cornstarch. In comparison, patients with GSD I generally require higher and more frequent doses (eg, 1.6-2.5 g/kg every 3-5 hours) due to impairment in both glycogenolysis and gluconeogenesis. 29 In our population, cornstarch intake ranged from 9 to 193 g/day, equivalent to approximately 30 to 700 kcal/day. This low dose and slower digestion of uncooked cornstarch compared with cooked starch, may explain the lack of correlation between cornstarch intake and the degree and variability of Glc 4 excretion.
A higher variability in Glc 4 excretion was associated with increased Glc 4 concentrations, but was not consistently observed in repeat collections by the same subject. The reason for this variability is unknown. In addition to the potential for dietary influences, physical activity may be a factor. 14 Twenty-four hour urine collections are considered a "gold standard" method for assessing urinary biomarker excretion. 30 However, 24-hour collections impose a significant burden on subjects and a risk for collection errors, 31 as demonstrated by the high rejection rate (22%) in our study. Appropriate storage of urine is another challenge. It was proposed that Glc 4 might be unstable at ambient temperature in some urine samples because of bacterial degradation, resulting in specimens with unexpectedly low concentrations. 32 Our studies indicate that Glc 4 is usually stable in urine for 1 week in a climate-controlled environment at ambient temperature (Supplemental Table 2). However, storage and transport of samples at 4 C or colder is recommended to ensure sample integrity. Several studies have demonstrated the equivalence of spot urines and 24-hour collections for a number of analytes normalized to creatinine. 33,34,35 Our results support the use of spot urines to evaluate Glc 4 in patients with GSD III.
The reliance on patients to comply with the 24-hour urine collection protocol and the prescribed dietary cornstarch therapy in an unsupervised setting was a limitation of this study. The reliability of the 24-hour collections accepted for analysis is supported by several lines of evidence: (a) the Glc 4 excretion rate (median: 45 mg/24 hours, range: 6-720) was comparable to published rates in patients with GSD III (9-45 mg/24 hours) 14 ; (b) total urine volume and creatinine excreted over 24 hours significantly increased with age and weight; and (c) the voiding frequency and urine output were comparable to a previous study. 28 To conclude, spot urines are generally reliable for assessing Glc 4 excretion in GSD III. Increased variability in Glc 4 excretion may be observed in patients who excrete higher Glc 4 concentrations. This variability does not appear to be caused by cornstarch intake. Repeat assessments in spot urines collected close together in time are advisable, to assess baseline Glc 4 concentrations and monitor trends in response to therapies in GSD III.