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Changes of plasma cGP/IGF‐1 molar ratio with age is associated with cognitive status of Parkinson disease

Abstract Objective Cognitive impairment is a common feature of Parkinson disease (PD), for which age is a major contributing factor. Insulin‐like growth factor‐1 (IGF‐1) declines with age and contributes to age‐related cognitive impairment in PD. Cyclic glycine‐proline (cGP) is a metabolite of IGF‐1 and normalizes bioavailable IGF‐1. Plasma cGP/IGF‐1 molar ratio that represents bioactive IGF‐1 in circulation, may associate with the cognitive status in PD. Methods We examined the association of plasma cGP/IGF‐1 molar ratio with the cognitive scores or age in PD patients with normal cognition (PD‐N, n = 74), mild cognitive impairment (PD‐MCI, n = 71), or dementia (PD‐D, n = 33), and with the cognitive scores in 23 age‐matched healthy controls. Plasma concentrations of IGF‐1, IGF binding protein‐3, and cGP were evaluated using enzyme‐linked immunosorbent assay (ELISA) and high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS), respectively. Results The cGP/IGF‐1 molar ratio was positively correlated with the age of PD‐N group, negatively correlated with the age of PD‐D group, and not associated with the age of PD‐MCI group. Independent of age, the cGP/IGF‐1 molar ratio was positively correlated with the cognitive scores of healthy controls, but not in PD groups. Conclusion Old healthy people with a higher cGP/IGF‐1 molar ratio showed better preserved cognition, possibly due to improved IGF‐1 function. Increased cGP/IGF‐1 molar ratio with age may contribute to cognitive retention in the PD‐N group. The absence or reversal of such association with age in the PD‐MCI and PD‐D groups may indicate the conversion of cognitive status in PD, if confirmed through longitudinal investigations within the individuals with advancing cognitive impairment.


BACKGROUND
Vascular health is critical for maintaining normal cognition in humans. 1,2 Functional plasma insulin-like growth factor-1 (IGF-1) plays an essential role in vascular remodeling of the adult brain 3 and supports the retention of normal cognition. 4 Plasma IGF-1 declines with age, 5 which is a major contributing factor to cognitive impairment in old people those are 60 and older. [4][5][6][7] Given that Parkinson disease (PD) is an age-related neurological condition, the decline of IGF-1 with age might contribute to cognitive decline in PD. 5,[8][9][10] Compared with the normal aging population, patients with PD have a higher risk of developing cognitive impairment, 11 and those with mild cognitive impairment (PD-MCI) have a higher risk of developing dementia (PD-D) than PD patients with normal cognition (PD-N). 12,13 Poor cognition in patients with PD is a recognized primary problem affecting patient and carer well-being. 14,15 A reliable plasma biomarker for predicting and tracking the changes in cognitive status may assist in identifying patients at greater risk of developing dementia and the window of opportunity for suitable intervention.
Plasma concentration of IGF-1 has been under clinical evaluation as a potential biomarker for IGF-1 function and its association with cognitive status of PD. 8,16 However, the findings have been mixed and difficult to interpret 8,16 because the majority of plasma IGF-1 is not bioavailable. The majority of plasma IGF-1 is bound to IGF-binding proteins (IGFBP)-3. This high-affinity binding prevents IGF-1 from activating its functional receptors. 17 Alternatively, the ratio of IGF-1/IGFBP-3 has been evaluated as a marker of the amount of unbound bioactive IGF-1. 17 Although the IGF-1/IGFBP-3 ratio may help to identify functional plasma IGF-1, its does not have clear association with cognitive function. 18 Our recent clinical and experimental studies suggest that the relative concentration of cyclic glycine-proline (cGP) to IGF-1 (cGP/IGF-1 molar ratio) represents the amount of bioavailable IGF-1 in plasma and has been shown to be a reliable biomarker of functional IGF-1 in circulation. [19][20][21][22] As a stable metabolite from a major binding site of IGF-1, 23 cGP retains the binding affinity to IGFBP-3 and effectively competes with IGF-1 to bind to plasma IGFBP-3, and it does so, in a concentration-dependent manner. 19 A higher concentration of plasma cGP would occupy a greater proportion of IGFBP-3 and increase the amount of free, bioavailable IGF-1. [19][20][21][22] To determine the potential association between plasma cGP/IGF-1 molar ratio and cognitive status, this cross-sectional study examined plasma concentrations of IGF-1, cGP, and IGFBP-3, and their ratios in PD patients with normal cognitive function, MCI, and dementia.

Study population and design
The study was approved by the Southern Health and Disability Ethics ( Figure 1). Healthy control participants (n = 24) were recruited from the local community. Twenty-six PD patients were excluded from the study due to incomplete cognitive assessments (n = 24) and the subsequent diagnosis of atypical symptoms (n = 2). One control participant was excluded due to an MCI classification. Plasma samples and neurological and cognitive assessments were available for 23 control participants, 74 PD with normal cognitive (PD-N), 71 PD with mild cognitive impairment (PD-MCI), and 33 PD with dementia (PD-D, Figure 1). All participants took their usual medications on the day of testing to allow for optimal performance. All testing was performed in the morning.

Inclusion criteria
Patients with PD included in the study were between age 50 and 90 years of age and meeting UK Brain Bank criteria 24 for idiopathic PD as confirmed by a movement disorders neurologist (T.J.A.). Patients with cognitive impairment exhibited motor features of PD for at least 12 months before the onset of cognitive impairment.

Exclusion criteria
Participants were excluded if there was a history of central nervous system (CNS) disorders other than PD (eg, stroke or head injury), any

Assessments of cognitive status
Cognitive status was determined using a battery of assessments meeting the requirements of the movement disorder society-task force (MDS-TF) Level II criteria. 24

Plasma sample collection and preparation
Non-fasting venous blood was collected in the morning using lithium heparin tubes (Becton Dickinson) and gently inverted as per manufacture directions. Filled tubes were placed on wet ice and immediately transferred to the processing laboratory. The samples were spun in a cooled centrifuge (4 • C) for 10 minutes at 2000g and plasma stored as 250 µL aliquots at −80ۤ • C until assayed.

Plasma extraction of cGP
The methodology of extraction of cGP from plasma has been described previously. 20,21 The cGP-1,5,6,7,8-13 C, 4-15 N (cGP-4x 13 provided an internal standard for the cGP assay. Briefly, cGP-4x 13 C, 1 × 15 N (50 µL of 500 ng/mL) was mixed with 100 µL of plasma, and then transferred to a Phree phospholipid removal cartridge (Phenomenex, Auckland, New Zealand). Five hundred milliliters of 1% formic acid in acetonitrile was added to the cartridge and centrifuged at 284g for 5 minutes at 4 • C. The filtrate was dried using a vacuum concentrator.
The dried samples were reconstituted in 100 µL 10% methanol/water (v/v) and transferred to an ultrapressure liquid chromatography vial for quantitation, and then centrifuged at 142g for 4 minutes at 4 • C to sediment any remaining particulates. Standards were prepared by spiking cGP into charcoal-stripped human plasma. Quality control samples, with cGP at two different concentrations (Bachem, G-1720), were utilized and then subjected to the same extraction procedure as the samples.

High-performance liquid chromatography mass spectrometry (HPLC-MS)
The  scores were analyzed in control and PD groups using multiple linear regression analysis after being adjusted for motor scores and age. The association between biological measures with age were analyzed in the controls and PD groups using multiple linear regression after being adjusted for motor and/or cognitive scores. As a secondary analysis, the association of biological measures with age was compared between the PD groups using multiple linear regression analysis. The significance level was set at P < 0.05. Data are presented as mean ± standard error of the mean (SEM) unless otherwise stated. Table 1 shows the demographic, clinical, and biological differences between the control and PD-N groups and within the PD groups. There was no difference in the age between the controls and combined PD groups, although the PD-N group was younger than the control (P <  Parametric data were analyzed using t test and one-way ANOVA with Tukey post hoc tests for comparison between controls and PD-N and within PD groups, respectively, and presented as mean (SEM). Non-parametric data were analyzed using Mann-Whitney U test and Kruskal-Wallis test with Dunn-Bonferroni post hoc test accordingly and presented as median (range). The gender effects were analyzed using chi-square test. * P < 0.05, ** P < 0.01, *** P < 0.001 indicating the differences from PD-N group; † P < 0.05, † † P < 0.01 indicating the difference from PD-MCI.

RESULTS
Plasma IGF-1 and IGFBP-3 concentrations were not associated with the age of control group after being adjusted for the cognitive scores.

DISCUSSIONS
Our findings suggest that old healthy people with a higher cGP/IGF-1 molar ratio have better preserved cognition independent of age, possibly due to more bioavailable IGF-1 in circulation. The cGP/IGF-1 molar ratio increased with age in the PD-N. However, such an association with age was absent in the PD-MCI and reversed to a decrease in the cGP/IGF-1 molar ratio with age in the PD-D. These age-related TA B L E 2 Association between biological measures and cognitive scores after being adjusted for age and/or motor scores in the control and PD groups  The changes of circulating IGF-1 and IGFBP-3 have been under clinical evaluations for their potential association with PD and their cognitive status with inclusive and inconsistent outcomes. 8,16,31,32 It is known that plasma IGF-1 concentration decreases with age, 5,18 whereas it increases in PD. 31 (Table 2), whereas they were associated with age in the PD groups independent of cognitive and motor function (Table 3).
Better preserved cognitive function of old healthy people was associated with higher plasma concentration of cGP and/or cGP/IGF-1 molar ratio, which was independent of age. The data support a role for cGP in maintaining cognitive function during brain aging. The effects of cGP in memory have been reported previously in rats. 22,29,30 For example, maternal administration of cGP improves learning and memory in young male rats. 22 The plasma concentration of IGF-1 and molar ratio of IGF-1/IGFBP-3 have been under clinical evaluation for predicting the cognitive function of old people with inconsistent outcomes. 4,6,7,18 Our data did not suggest the relationship between cognitive scores and IGF-1 concentration and IGF-1/IGFBP-3 molar ratio in normal controls (Table 3). Thus cGP concentration and cGP/IGF-1 molar ratio may be more closely related to the cognitive function of healthy old people.
Such an association with cognitive function was absent in PD groups, possibly due to their lower cognitive scores and abnormal cognitive function. Although the cognitive function of PD-N was "normal," the group had significant lower scores when compared to the healthy control group. This may suggest an initial decline of cognition in the PD-N due to accelerated brain aging in PD. Age-related decline of IGF-1 is a major contributor to cognitive impairment, including in PD. [4][5][6][7][8]16 Our results suggest that the biological measures of PD groups changed with age, which were dependent on the cognitive status (Tables 3 and 4, Figure 2).

TA B L E 3
Association between the biological measures and age after being adjusted for cognitive and/or motor scores in the control and PD groups  Plasma IGF-1 decreased with age of the PD-N group. As an autocrine response to improve the amount of bioavailable IGF-1 in plasma, [19][20][21][22] cGP correspondingly increased with age, leading to an age-associated increase of cGP/IGF-1 molar ratio in the PD-N group (Table 3 and Figure 2G). The increase of the cGP/IGF-1 molar ratio with age is a compensatory response to the age-related decline of IGF-1 function in circulation that may contribute to the preserved cognitive function in PD-N patients.
This age-related increase in cGP/IGF-1 ratio in the PD-N group was absent in the PD-MCI group (Table 3, Figure 2H), whereas it was statistically reversed to a decrease with age in the PD-D group (ie, lower ratio with greater age). This negative association was driven clearly by a progressive decline of cGP with age (Tables 3 and 4, Figure 2F). Low plasma concentration of cGP and/or the cGP/IGF-1 molar ratio has been suggested to be associated with impairment of the autocrine regulation of IGF-1 in hypertension 21 and stroke patients, 20 and may also contribute to cognitive impairment in PD. Indeed, the administration of cGP can prevent ischemic brain injury, 19,33 normalize blood pressures, 34 and improve memory in rats. 22 A longitudinal study shows that the cGP/IGF-1 molar ratio increases while patients recover from stroke and that the cGP/IGF-1 molar ratio at hospital admission (<3 days of stroke) predicts 3 months recovery and clinical outcome of stroke. 20 These age-related observations in PD need to be validated through longitudinal studies in individuals with advancing cognitive impairments. If confirmed, the changes of cGP/IGF-1 molar ratio with age may potentially be used as a biomarker for assisting the diagnosis and/or prognosis of cognitive risk and conversions of PD.
Poor vascular health, like hypertension, is a common risk factor contributing to cognitive impairment in PD and stroke patients. [35][36][37] The function of plasma IGF-1 is essential for cerebral vascular remodeling of mature brains and is mediated through the blood face of cerebral vessels. 3 The administration of cGP protects neuronal and, more so, vascular injury from ischemia through promoting IGF-1-mediated vascular protection/remodeling in rats. 19 Given the nature of cerebral vascular degeneration/dysfunction in age-related cognitive impairment, 38 the changes in the plasma cGP/IGF-1 molar ratio may represent IGF-1-mediated cerebral vascular function/remodeling and its association with cognitive function. Given the role of cGP in memory, 22 we speculate that intervention with cGP may restore the cGP/IGF-1 molar ratio and improve the cognitive function in PD patients with progressive decline of plasma cGP and/or cGP/IGF-1 molar ratio.
The small number of control participants and PD-D patients used for analysis is a limitation of the study. However, the publication of this hypothesis-generating finding will promote research for confirming the hypothesis through larger and longitudinal studies.

CONCLUSION
An increase in plasma cGP/IGF-1 molar ratio with age may contribute to the preserved cognitive function in the PD-N group. In contrast, the decrease in the ratio with age may be associated with dementia in the PD-D group. The static relationship between cGP/IGF-1 molar ratio and age in the PD-MCI group could be a transition phase before dementia. These observations raise the possibility that the association between cGP/IGF-1 ratio and age may assist prediction of cognitive status and risk of advancing cognitive impairment in patients with PD if further confirmed in longitudinal research.

ACKNOWLEDGMENTS
We would like to acknowledge the Cancer Society Tissue Bank for providing the bio banking services, and Leslie Livingston, Sophie Grenfell, and Bob Young from New Zealand Brain Research Institute, New Zealand, for completing the clinical and cognitive assessments.

The clinical investigation and blood collection was funded by Health
Research Council New Zealand and Lottery Health New Zealand. The biological analysis was funded by Auckland Medical Research Foundation, New Zealand.

CONFLICTS OF INTEREST
The authors have no conflicts of interest to disclose.

AUTHOR CONTRIBUTIONS
Dawei Fan: biological assays, statistical analysis, and manuscript preparation; Toni Pitcher: sample collection and management and manuscript revision; Michael MacAskill: data management and statistical analysis; John Dalrymple-Alford: cohort PI and manuscript revision; Tim Anderson: cohort PI and manuscript revision; and Jian Guan: project PI, conception, and writing of the manuscript.