life

Article
Utility of Fasting C-Peptide for the Diagnostic Differentiation of
Patients with Type 1, Type 2 Diabetes, MODY, and LADA
Ricardo Alemán-Contreras 1,† , Rita A. Gómez-Díaz 2,† , Maura E. Noyola-García 1 , Rafael Mondragón-González 2 ,
Niels Wacher 2 and Aldo Ferreira-Hermosillo 3, *

1 Servicio de Medicina Interna, Hospital de Especialidades, Centro Médico Nacional Siglo XXI,
Instituto Mexicano del Seguro Social, Mexico City 06720, Mexico; [email protected] (R.A.-C.);
[email protected] (M.E.N.-G.)
2 Unidad de Investigación Médica en Epidemiología Clínica, Hospital de Especialidades,
Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City 06720, Mexico;
[email protected] (R.A.G.-D.); [email protected] (R.M.-G.); [email protected] (N.W.)
3 Unidad de Investigación Médica en Enfermedades Endocrinas, Hospital de Especialidades,
Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City 06720, Mexico
* Correspondence: [email protected]; Tel.: +52-5556276900 (ext. 21913)
† These authors contributed equally to this study.

Abstract: Background: The prevalence of obesity has increased in patients with type 1 diabetes (T1D)
and latent autoimmune diabetes of the adult (LADA), limiting the use of clinical features such as
the body mass index for its differentiation with type 2 diabetes (T2D). Additionally, some patients
with maturity-onset diabetes of the young (MODY) or LADA are misdiagnosed as having T2D. The
evaluation of autoantibodies and genetic testing are not fully available. We aimed to evaluate the
utility of a widely available and less expensive diagnostic tool such as C-peptide to differentiate
between T1D, T2D, MODY, and LADA. Methods: Our study included 38 patients with T1D, 49 with
Citation: Alemán-Contreras, R.; T2D, 13 with MODY, and 61 with LADA. We recorded anthropometric measurements, biochemical
Gómez-Díaz, R.A.; Noyola-García, profiles, and antidiabetic treatment and determined C-peptide, anti-GAD65, and anti-IA2 antibodies.
M.E.; Mondragón-González, R.; Results: C-peptide concentration differed significantly among populations (T1D: 0.2 ng/mL; T2D:
Wacher, N.; Ferreira-Hermosillo, A. 2.4 ng/mL; MODY: 1.14 ng/mL; LADA: 1.87 ng/mL). Through a ROC curve, we observed that
Utility of Fasting C-Peptide for the the C-peptide cut-off point of 0.95 ng/mL allows differentiation between T1D and T2D (sensitivity
Diagnostic Differentiation of Patients 82%, specificity 77%); 0.82 ng/mL between T1D and LADA (sensitivity 82%, specificity 77%); and
with Type 1, Type 2 Diabetes, MODY,
1.65 ng/mL between T2D and MODY (sensitivity 72%, specificity 72%). Conclusions: C-peptide is
and LADA. Life 2024, 14, 550. https://
useful for the diagnostic differentiation of patients with type 1, type 2 diabetes, MODY, and LADA.
doi.org/10.3390/life14050550

Academic Editors: Fabrizio Keywords: autoimmunity; C-peptide; MODY; type 1 diabetes; type 2 diabetes
Montecucco, Ionela Lacramioara
Serban, Daniela Maria Tanase and
Mariana Floria

Received: 22 February 2024

1. Introduction
Revised: 4 April 2024 According to the Encuesta Nacional de Salud y Nutrición 2022 (National Health and
Accepted: 19 April 2024 Nutrition Survey 2022), the prevalence of people diagnosed with diabetes mellitus > 20 years
Published: 25 April 2024 of age in Mexico is 12.6% [1]. However, the prevalence of type 1 diabetes (T1D) and other
types of diabetes, such as latent autoimmune diabetes of the adult (LADA), which is charac-
terized by late development of autoimmunity [2], or Maturity Onset Diabetes of the Young
(MODY) [3], in Mexico is unknown.
Copyright: © 2024 by the authors.
In the clinical setting, age, and body mass index (BMI) are widely used to guide the
Licensee MDPI, Basel, Switzerland.
diagnosis of the type of diabetes a patient has, but they do not detect autoimmunity [4]. This
This article is an open access article
leads to misdiagnosis and the use of inadequate treatments, such as oral antidiabetics in a
distributed under the terms and
patient with T1D or high doses of insulin instead of the administration of oral antidiabetics
conditions of the Creative Commons
in some patients with T2D or MODY. This difficulty in diagnosis is increased in the case of
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
youths with overweight/obesity [5]. Because of this, some studies have been proposed to
4.0/).
differentiate between patients with and without autoimmunity, such as the determination

Life 2024, 14, 550. https://doi.org/10.3390/life14050550 https://www.mdpi.com/journal/life

Life 2024, 14, 550 2 of 13

of autoantibodies or the search for risk haplotypes in the human leukocyte antigen (HLA)
complex, analogous to the major histocompatibility complex (MHC) [6]. However, these
studies are expensive, and their availability is limited.
Due to the above, other proposals have arisen for diagnostic differentiation, such as
the determination of C-peptide concentrations [7]. The presence of C-peptide is associ-
ated with the degree of pancreatic ß-cell reserve, so a patient with T1D has undetectable
concentrations after a period of 10 years, while the pediatric population recently diag-
nosed or patients with T2D or MODY persist with measurable secretion of this peptide [8].
Furthermore, patients with LADA show a lower concentration in comparison with T2D [9].
The objective of this study was to assess the utility of C-peptide for diagnostic differ-
entiation between T1D, T2D, MODY, and LADA in the adult Mexican population and to
evaluate its correlation with clinical parameters.

2. Materials and Methods

A cross-sectional analytical study was performed on consecutive patients previously
diagnosed with T1D, T2D, LADA, or MODY attending the Diabetes Clinic at the Hospital
de Especialidades of Centro Médico Nacional Siglo XXI, a tertiary referral center. This
study was approved by the Local Research and Ethics in Health Research Committee
(F-2016-3601-3). We included patients that attended at least three visits in the last year, aged
18–70 years, with previous clinical diagnosis of T1D, T2D, or MODY, classified according to
the standards of the American Diabetes Association (ADA) [10], or with LADA, according
to the criteria of the Immunology of Diabetes Society [2]. Patients with T1D were diagnosed
during childhood with the presence of an islet autoantibody and an immediate requirement
for insulin (in comparison with patients with LADA who could be treated with oral
antidiabetic drugs for at least 6 months). We included patients clinically diagnosed with
HNF1A-MODY. Those patients had an increase in serum glucose > 90 mg/dL in the
oral glucose tolerance test, the presence of glycosuria when serum glucose was lower
than 180 mg/dL in several follow-up tests, a normal concentration of HDL-cholesterol
(HDL-c), and an initial sensitivity to sulfonylurea drugs with insulin requirement after a
year. Furthermore, those patients were assessed through the MODY probability calculator
available at www.diabetesgenes.org (accessed on 1 February 2024) [11], that is based on
the prediction model performed by Shields et al. that detects the chance of having MODY
when the probability cutoff is >25% [12]. For improving the detection capacity of the
calculator, we applied the corrected cutoff proposed by Silva Santos et al. of >36% [13].
This cutoff increases the predictive positive value by 74.4% and the predictive negative
value by 73.5% [13].
This study included all the patients recruited from their diabetes appointments who
accepted participation. Their clinical diagnosis was verified through their medical records.
We excluded patients with other forms of diabetes. The objective of this study was explained
to all participants, who gave their written informed consent. The protocol was conducted
in accordance with the Declaration of Helsinki.

2.1. Anthropometric Measurements

During the office visit, age, sex, weight (kilograms), height (meters), and waist cir-
cumference (centimeters) were recorded. Only one of the investigators performed the
anthropometric measurements using a calibrated scale with a stadiometer and flexible,
non-expandable metric tape. The waist circumference (WC) was measured at the midpoint
between the last rib and the upper border of the anterosuperior iliac spine. BMI was
calculated as weight divided by the height squared, and the waist/height ratio (WHtR)
was calculated.

2.2. Biochemical Determinations

After an 8 h fast, a sample of 6 mL of blood was taken in a BD Vacutainer tube
(BD Franklin Lakes, NJ, USA) and centrifuged at 3150× g for 15 min to obtain serum.
Life 2024, 14, 550 3 of 13

Glucose, total cholesterol (TC), and triglycerides were determined using a commercial
kit (COBAS, Roche Diagnostics, Indianapolis, IN, USA) through photocolorimetry using
a Roche Modular P800 spectrophotometer (Roche Diagnostics, Indianapolis, IN, USA).
For the determination of HDL-c, an aliquot of serum was treated with polyethylenglicol
and dextran sulfate and analyzed with a commercial kit for cholesterol (COBAS, Roche
Diagnostics, Indianapolis, IN, USA). At the same time, 3 mL of blood was collected in a
BD Vacutainer tube with EDTA-K3 (BD Franklin Lakes, NJ, USA) for evaluation of Gly-
cated hemoglobin (HbA1c). HbA1c was evaluated through turbidometric immunoanalysis
(COBAS, Roche Diagnostics, Indianapolis, IN, USA). LDL-cholesterol (LDL-c) was calcu-
lated through the Friedewald formula: LDL-c (mg/dL) = TC mg/dL − (HDL-c mg/dL +
triglycerides mg/dL/5), whenever triglycerides were less than 400 mg/dL.

2.3. Determination of C-Peptides and Antibodies

Determination of C-peptide was performed with a COBAS commercial kit (Roche
Diagnostics, USA) in fresh serum (after an 8 h fast). The limit of detection of the test is
0.010–40 ng/mL, with a coefficient of inter-assay variation of 4.6% and intra-assay variation
of 5%. All values below the limits of detection were considered the minimum. The test
cross-reactivity with human insulin is 0.005%. Determination of antibodies anti-glutamate
descarboxylase 65 (anti-GAD65) and anti-tyrosine phosphatase related to islet 2 antigen
(anti-IA2) was performed through ELISA technique with a commercial kit, Medizym
Brand (Medipan GMBH, Blankenfelde-Mahlow, Germany). For anti-GAD65, the cut-off for
positivity is >5 IU/mL (specificity 98.6%, sensitivity 92.3%). The test analytical sensitivity
reported is 0.8 IU/mL and functional 4 IU/mL, with coefficients of inter-assay variation of
7% and intra-assay variation of 4%. For anti-IA2, the cut-off for positivity is >10 IU/mL
(specificity 98%, sensitivity 75%). The test analytical sensitivity reported is 0.5 IU/mL and
functional sensitivity is 0.8 IU/mL, with coefficients of inter-assay variation of 6% and
intra-assay variation of 5%.

2.4. Statistical Analysis

We evaluated normality using a Kolmogorov–Smirnov test. Quantitative variables
are described using medians with interquartile ranges due to the distribution of data.
Qualitative variables are described using frequency and percentages. The association
between qualitative variables was analyzed using the chi-squared test and an ANOVA if
there were more than two groups. Receiver operating characteristic (ROC) curves were
made of concentrations of C-peptide to identify the cut-off with optimum sensitivity and
specificity that would allow diagnostic differentiation, with 95% confidence intervals. We
also determined the area under the curve (AUC). The correlation between variables was
analyzed using the Spearman test. We evaluated all the clinical and biochemical factors
registered that could influence C-peptide concentration with a multiple regression analysis.
A p < 0.05 was considered statistically significant. For statistical analysis, statistical packages
SPSS version 20 and STATA version 11 were used.

3. Results
The study included 161 patients: 22.3% initially diagnosed with T1D, 30.4% with T2D,
9.3% with MODY, and 37.9% with LADA; 59% of the patients were women, with a median
age of 42 years (28–62 years). All patients have had more than 10 years since the diagnosis
of diabetes. Patients with T2D were older, followed by patients with LADA, MODY, and
finally patients with T1D. It was found that WC was greater in patients with T2D compared
with T1D, MODY, or LADA, while WHtR was different between the various populations.
BMI showed significant differences between patients with T1D and T2D, and LADA
and T2D.
When comparing T1D and MODY, differences were observed in the glucose (p = 0.028)
and C-peptide (p < 0.001) concentrations. When comparing MODY and LADA, only age
(p = 0.007) was different (Table 1). Fasting glucose concentrations in patients with MODY
Life 2024, 14, 550 4 of 13

(185 mg/dL) or LADA (190 mg/dL) were higher in comparison with those of patients
with T1D (138 mg/dL) or T2D (135 mg/dL), without differences between T1D and T2D.
C-peptide concentrations were significantly different between the different populations
(T2D vs. T1D p < 0.001; T2D vs. MODY p = 0.028; T1D vs. LADA p < 0.001; T2D vs.
LADA p = 0.019). There was no difference in C-peptide concentration between patients
with T1D and MODY and between MODY and LADA. In order to evaluate if hyper-
glycemia influenced C-peptide levels, we excluded subjects with glucose levels higher than
11.1 mmol/L: 2 patients with T2D, 7 patients with T1D, 5 patients with MODY, and
27 patients with LADA. Even after those patients were excluded, C-peptide concentrations
were significantly different among the groups (T2D vs. T1D p < 0.001; T2D vs. MODY
p = 0.036; T1D vs. LADA p < 0.001; T2D vs. LADA p = 0.010) (Table 2).

Table 1. Comparison of anthropometric and clinical variables between patients with type 1 diabetes,
type 2 diabetes, LADA, and MODY.

p **
T1D T2D MODY LADA
(n = 38 *) (n = 49) (n = 13 *) (n = 61) T2D vs. T2D vs. T1D vs. T2D vs. LADA vs.
T1D MODY LADA LADA MODY
27 60 37 50
Age (years) <0.001 <0.001 <0.001 0.010 0.007
(23–35) (46–71) (25–52) (43–59)
Disease duration 17 13.6 19 12
NS
(years) (11.1–24) (11–16.9) (11.2–30.4) (8.2–14.7)
Female (%) 66 53 54 61 NS
24.4 29 26.4 25.9
BMI (kg/m2 ) 0.012 NS NS 0.013 NS
(22.2–28.5) (24.9–31.7) (24.7–27.8) (24.0–28.4)
88 105 91 92
WC (cm) <0.001 0.012 0.012 <0.001 NS
(79–94) (92–114) (77–101) (87–99)
85 107 91 92
Female <0.001 NS 0.015 0.009 NS
(77–94) (90–113) (82–112) (87–97)
90 105 85 94
Male 0.008 0.010 NS 0.002 NS
(80–102) (94–116) (70–96) (86–100)
0.54 0.64 0.54 0.59
WHtR <0.001 0.014 0.001 0.002 NS
(0.49–0.59) (0.58–0.71) (0.46–0.60) (0.56–0.62)
0.53 0.66 0.57 0.59
Female <0.001 NS <0.001 0.008 NS
(0.48–0.58) (0.60–0.74) (0.50–0.72) (0.57–0.63)
0.55 0.62 0.48 0.57
Male NS 0.025 NS 0.019 NS
(0.49–0.62) (0.55–0.67) (0.42–0.55) (0.50–0.60)
T1D: type 1 diabetes; T2D: type 2 diabetes; LADA: Latent Autoimmune Diabetes in Adults; MODY: Maturity
Onset Diabetes of the Young; NS: not significant; BMI: body mass index; WC: waist circumference; WHtR: waist to
height ratio. The results of quantitative variables are given in median with interquartile range. * Two patients with
MODY were reclassified as T1D due to the positivity of anti-GAD. Results of quantitative variables are given in
median with an interquartile range. ** There were no statistical differences among patients with T1D vs. MODY.

The concentration of HbA1c, total cholesterol, triglycerides, LDL-c, HDL-c, and pro-
teins in 24 h urine showed no difference between kinds of diabetes, with the exception
of LDL-c in cases with LADA. The determination of anti-GAD 65 autoantibodies was
performed in the whole population: 55% of the patients with T1D had positive anti-GAD65
antibodies, compared with 4% (2/49) of the patients with T2D. Both patients were classified
as T2D due to their adequate glycemic control with oral antidiabetic drugs (HbA1c < 7%,
53 mmol/mol) despite disease duration. As for MODY, two patients presented positive
antibodies; however, in accordance with their clinical and biochemical characteristics, they
were re-classified as patients with T1D. Determination of anti-IA2 autoantibodies was
carried out in all patients, with 42% of the population with T1D and 62% of LADA patients
being positive. (Table 2).
Life 2024, 14, 550 5 of 13

Table 2. Comparison of biochemical and immunological variables between patients with type 1
diabetes, type 2 diabetes, LADA, and MODY.

p **
T1D T2D MODY LADA
(n = 38 *) (n = 49) (n = 13 *) (n = 61) T2D vs. T2D vs. T1D vs. T2D vs. LADA vs.
T1D MODY LADA LADA MODY
138 135 185 190
Glucose (mg/dL) NS 0.04 0.001 0.001 NS
(81–188) (115–176) (116–268) (139–254)
8.8 8.25 9.8 9.4
HbA1c (%) NS
(7.6–10.1) (6.7–10.2) (7.8–12.1) (7.2–11.0)
73 67 84 79
HbA1c (mmol/mol) NS
(60–87) (50–88) (62–109) (55–97)
188 181 207 195
TC (mg/dL) NS
(144–223) (161–211) (163–222) (162–233)
133 164 156 172
TG (mg/dL) NS
(89–198) (120–234) (132–280) (117–222)
110 99 96 128
LDL-c (mg/dL) NS NS 0.036 0.002 NS
(79–133) (76–123) (85–147) (94–154)
HDL-c (mg/dL) 45 (39–61) 45 (34–56) 41 (33–52) 47 (41–54)
Female 52 (43–64) 49 (42–60) 40 (31–57) 48 (42–54) NS
Male 39 (34–52) 39 (30–51) 41 (33–55) 46 (40–53)
Creatinine clearance 102
77 (60–95) 91 (72–140) 92 (77–112) 0.035 NS NS 0.003 NS
(mL/min/24 h) (62–126)
0.76 0.91 0.70 0.75
Creatinine (mg/dL) 0.033 NS NS 0.007 NS
(0.64–0.96) (0.73–1.26) (0.64–0.95) (0.64–0.93)
0.2 2.4 1.14 1.87
C-Peptide (ng/mL) <0.001 0.028 <0.001 0.019 NS
(0.01–0.85) (1.3–3.6) (0.80–1.83) (1.27–2.48)
C-peptide after
0.2 2.69 1.39 1.72
hyperglycemic <0.001 0.036 <0.001 0.010 NS
(0.01–0.95) (1.3–3.6) (0.77–2.0) (0.80–2.30)
exclusion
Anti-GAD+ 55% 4% 0% 23%
0.001 NS 0.037 0.050 NS
(n = 161) (21/38) (2/49) (0/13) (14/61)
42% 0% 0% 62%
Anti-IA2 + (n = 161) 0.007 NS <0.001 <0.001 <0.001
(16/38) (0/49) (0/13) (38/61)
T1D: type 1 diabetes; T2D: type 2 diabetes; LADA: Latent Autoimmune Diabetes in Adults; MODY: Maturity
Onset Diabetes of the Young; NS: not significant; HbA1c: glycosylated hemoglobin A1c; TC: total cholesterol; TG:
triglycerides; LDL-c: low density lipoprotein cholesterol; HDL-c: high density lipoprotein cholesterol; anti-GAD:
glutamic acid descarboxylase antibodies; anti-IA2: tyrosine phosphatase antibodies. * Two patients with MODY
were reclassified as T1D due to the positivity of anti-GAD. The results of quantitative variables are given in the
median with an interquartile range. ** There were no statistical differences among patients with T1D vs. MODY,
except for glucose concentration (p = 0.028) and C-peptide (p < 0.001).

Table 3 shows the antidiabetic treatment for the study groups. More than half of
the patients in each group were under treatment with insulin, in response to the lack of
metabolic control noted (see Table 2). It is remarkable that patients with MODY received
more oral antidiabetic treatment in addition to insulin (77% received sulfonylurea and/or
54% metformin, with 69% receiving insulin). There were no differences in C-peptide levels
between patients with (n = 31) or without (n = 130) sulfonylurea treatment (1.35 ng/mL
[0.75–2.23] vs. 1.71 ng/mL [0.67–2.71], respectively; p = 0.764).
The cut-off for fasting C-peptide was 0.95 ng/mL for diagnostic differentiation be-
tween T1D and T2D (sensitivity 82%, specificity 77%, AUC 0.88; Figure 1A); the cut-off for
fasting C-peptide was 0.82 ng/mL for diagnostic differentiation between T1D and LADA
(sensitivity 82%, specificity 77%, AUC 0.86; Figure 1B); and the cut-off for fasting C-peptide
was 1.65 ng/mL for diagnostic differentiation between T2D and MODY (sensitivity 72%,
specificity 72%, AUC 0.75).
 Medication (T2D vs. (T2D vs. (T1D vs. (T2D
(n = 38) (n = 49) (n = 13) (n = 61)
T1D) MODY) LADA) vs. LADA)
Rapid-acting insulin 29 9 3 12
<0.001 NS <0.001 NS
(Humalog or lispro) (76%) (18%) (23%) (20%)
Life 2024, 14, 550 6 of 13
Intermediate-acting 14 24 4 25
0.001 NS NS NS
insulin (NPH) (37%) (49%) (31%) (41%)
Long-acting insulin 21 5 4 10
Table 3. Use of diabetes medication by all subjects and 0.001 NSdiabetes
those based on <0.001
type. NS
(glargine) (55%) (10%) (31%) (16%)
Metformin 5T1D(13%) 27
T2D
(45%) MODY 7 (54%) LADA42 (69%) p 0.001 p NS <0.001
p NS
p
Medication (T2D vs. (T2D vs. (T1D vs. (T2D
Sulphonylurea 0 (0%)
(n = 38) 4 (8%)
(n = 49) 10
(n = 13)(77%) 17
(n = 61) (28%) NS <0.001 <0.001 0.006
T1D) MODY) LADA) vs. LADA)
Use of insulin 100% 29 (59%) 9 (69%) 33 (54%) <0.001 <0.001 <0.001 NS
Rapid-acting insulin 29 9 3 12
(Humalog or lispro) 0.80 0.50 0.61 0.12 <0.001 NS <0.001 NS
(76%) (18%) (23%) (20%)
Insulin doses (U/kg) <0.001 NS <0.001 <0.001
Intermediate-acting (0.60–1.0)
14 (0.31–0.67)
24 (0.39–0.65)
4 25(0.04–0.51)
0.001 NS NS NS
insulin
Total (NPH)dose
insulin (37%)
46.5 (49%)35 (31%) 34 (41%) 31
Long-acting insulin 21 5 4(27–55) 10 (18–42) 0.002 NS <0.001 NS
(U/day) (38–70) (25–48.5) 0.001 NS <0.001 NS
(glargine) (55%) (10%) (31%) (16%)
X2 or Mann-Whitney U, according to the type of variable. p < 0.05 was considered significant. T1D:
Metformin 5 (13%) type 127 (45%) T2D:7 type
diabetes; (54%)2 diabetes.
42 (69%) 0.001 NS <0.001 NS
Sulphonylurea 0 (0%) 4 (8%) 10 (77%) 17 (28%) NS <0.001 <0.001 0.006
Use of insulin 100% The cut-off for
29 (59%) fasting C-peptide
9 (69%) 33 (54%) was <0.001
0.95 ng/mL <0.001for diagnostic differentiation
<0.001 NS be-
0.80 tween T1D
0.50 and T2D (sensitivity
0.61 82%,
0.12 specificity 77%, AUC 0.88; Figure 1A); the cut-off for
Insulin doses (U/kg) <0.001 NS <0.001 <0.001
(0.60–1.0)fasting
(0.31–0.67) (0.39–0.65) (0.04–0.51)
C-peptide was 0.82 ng/mL for diagnostic differentiation between T1D and LADA
Total insulin dose 46.5 (sensitivity 35 82%, specificity
34 77%, 31AUC 0.86; Figure 1B); and the cut-off for fasting C-pep-
(U/day) 0.002 NS <0.001 NS
(38–70) tide was(25–48.5)
1.65 ng/mL(27–55) (18–42)differentiation between T2D and MODY (sensitivity
for diagnostic
X2 or Mann-Whitney
72%, U, according
specificity 72%, to the type of variable. p < 0.05 was considered significant. T1D: type 1 diabetes;
AUC 0.75).
T2D: type 2 diabetes.

Figure 1. ROC curve for fasting C-peptide: (A) for diagnosis between type 1 and type 2 diabetes;
Figure 1. ROC curve for fasting C-peptide: (A) for diagnosis between type 1 and type 2 diabetes; (B)
(B) for diagnosis between type 1 diabetes and LADA.
for diagnosis between type 1 diabetes and LADA.
It was observed that anti-GAD65 autoantibodies for the diagnosis of T1D have a
It was observed
sensitivity that anti-GAD65
of 37%, specificity of 91%,autoantibodies for the
positive predictive diagnosis
power (PPP)ofof
T1D71%,have a sen-
negative
sitivity of 37%, specificity of 91%, positive predictive power (PPP) of 71%,
predictive power (NPP) of 72%, false positives of 8%, and diagnostic certainty of 79%. negative pre-
dictive power (NPP) of 72%, false positives of 8%, and diagnostic certainty
Likewise, anti-IA2 autoantibodies have a sensitivity of 19%, specificity of 100%, PPP of of 79%. Like-
wise,
100%,anti-IA2 autoantibodies
NPP of 69%, and certaintyhaveof a87%
sensitivity of 19%, specificity
for the diagnosis of T1D. of 100%, PPP of 100%,
NPP As of 69%,
shownandincertainty
Table 4, of 87% for the
a moderate diagnosiswas
correlation of T1D.
observed between C-peptide and
As shown in Table 4, a moderate correlation was observed
WC in the total study population and in patients with T2D. Likewise, betweeninC-peptide anda
this group,
WC in the total study population and in patients with T2D. Likewise, in
positive association was found with triglycerides and a negative correlation with HDL-c. this group, a
positive association was found with triglycerides and a negative correlation
In addition, a correlation was observed between C-peptide and weight in the total study with HDL-c.
In addition, as
population, a correlation was observed
well as in patients with T1D between C-peptide and weight in the total study
and T2D.
population, as well
A multiple as in patients
regression analysiswithwasT1D and T2D.
performed (Table 5), where 33% of C-peptide con-
centration was influenced by WC (ß = 0.468), insulin use (ß = −0.280), and sex
(ß = 0.217). Upon dividing by groups, in T2D, 39% of C-peptide concentration was influ-
enced by WC (ß = 0.496), sex (ß = 0.307), and insulin use (ß = −0.288), while in T1D, 19% of
C-peptide concentration was influenced by BMI (ß = 0.482). In LADA, HbA1c (ß = −0.280)
influenced 6% of C-peptide concentration. In MODY, C-peptide was not influenced by
any studied variable. Neither age, diabetes duration, oral treatment used, nor creatinine
clearance influenced C-peptide concentrations. Evaluating alternative regression models,
we also corroborate that none of the other variables influenced C-peptide concentration.
Life 2024, 14, 550 7 of 13

Table 4. C-peptide correlation with anthropometric and biochemical parameters.

Total Study
Type 1 Diabetes Type 2 Diabetes MODY LADA
Population
rho p rho p rho p rho p rho p
Fasting
C-peptide
vs. weight 0.452 <0.001 0.524 0.012 0.499 0.001 0.446 NS 0.276 0.039
vs. BMI 0.390 <0.001 0.454 0.034 0.380 0.017 0.410 NS 0.317 0.019
vs. waist cir-
0.491 <0.001 0.430 NS 0.506 0.001 0.237 NS 0.212 NS
cumference
vs. WHtR 0.397 <0.001 0.369 NS 0.332 0.039 0.213 NS 0.142 NS
vs. TAG 0.408 <0.001 0.272 NS 0.507 0.001 0.005 NS 0.372 0.006
vs. HDL-c −0.295 0.001 −0.403 NS −0.326 0.049 −0.339 NS −0.299 0.028
MODY: Maturity-Onset Diabetes of the Young: NS: not significant; BMI: body mass index; WHtR: waist-to-
height ratio; HbA1c: glycosylated hemoglobin A1c; TC: total cholesterol; TG: triglycerides; HDL-c: high-density
lipoprotein cholesterol.

Table 5. Multiple regression analysis for C-peptide concentrations.

Variable Total Group Type 2 Diabetes Type 1 Diabetes LADA

ß * (IC95%) p ß * (IC95%) p ß * (IC95%) p ß * (IC95%) p
0.468 0.496
WC (0.038–0.074) <0.001 (0.033–0.107) <0.001 NS NS
−0.280 −0.288
Insulin use (−1.351–−0.401) <0.001 (−2.093–−0.110) 0.030 NS NS
0.217 0.307
Sex (0.207–1.178) 0.006 (0.183–2.211) 0.022 NS NS
0.482
BMI NA NS (0.012–0.235) 0.031 NS
−0.280
HbA1c NS NS NS (−0.227–−0.002) 0.047
WC: waist circumference; BMI: body mass index; HbA1c: glycosylated hemoglobin A1c. * Adjusted by sex, age,
waist circumference, BMI, insulin use, HbA1c, fasting glucose, metformin, and sulphonylurea use.

4. Discussion
Type 1 diabetes was once considered the only form of diabetes in children and adoles-
cents; however, there is a growing prevalence of T2D in that population due to increased
overweight and obesity [14]. This situation has generated difficulty in the adequate classi-
fication of a patient diagnosed with diabetes, where one may face a patient with obesity,
which generates insulin resistance (which orients us towards a diagnosis of T2D) [5] or a
patient with autoimmunity that develops obesity due to over-insulinization and an inade-
quate diet (which directs us towards a patient with T1D with metabolic syndrome, also
known as “double diabetes”) [15]. On the other hand, there are also patients without a
history of insulin resistance that debut with insulin treatment-resistant hyperglycemia at
different ages (which can orient us towards a diagnosis of MODY) [16] and patients that
display a milder autoimmune process, slower ß-cell failure, and insulin independence for
6 to 12 months after diagnosis (which orient us towards a diagnosis of LADA) [17]. This
entity is not rare, since it is estimated that almost 10% of patients diagnosed with T2D have
LADA [18]. However, its clinical differentiation has become difficult due to overweight
and adiposity, which are also risk factors for LADA [18].
One way to differentiate between patients with and without autoimmunity is the
determination of autoantibodies [19]. Nevertheless, the detection of a single autoantibody
Life 2024, 14, 550 8 of 13

is insufficient [20], and there is a possibility of not detecting them at the blood level (for
example, in a patient with idiopathic T1D) [6]. Additionally, as observed in our study, the
prevalence of antiGAD65 autoantibodies has been reported to be 4.3% [21] in patients with
T2D. This might be related to the frequency of those autoantibodies even in the non-diabetic
population (0.7–4.8%) [22].
Considering those difficulties, there is a need to use other biochemical resources
that allow the adequate differentiation of patients with T1D, T2D, MODY, and LADA.
C-peptide has been a useful tool in the adult population since patients with T1D show
deficient levels of C-peptide 2 or 3 years after diagnosis, while patients with T2D or MODY
persist with detectable levels [23]. In addition, there is the advantage of broad commercial
availability, comparatively low cost, and easy availability [24]. For its determination, C-
peptide requires immediate lab analysis due to its quick degradation [25]. Those conditions
were considered for C-peptide quantification in our protocol. Regarding the different
assays for its determination, C-peptide could also be quantified after 8 to 10 h of fasting
or after stimulation with glucagon, intravenous/oral glucose, tolbutamide, sulfonylurea,
or a mixed meal [26], but these techniques are not effective in the presence of insulin
treatment. Despite the fact that fasting C-peptide has been criticized due to its limited
ability to detect subtle levels, other studies have proven that it correlates with urinary
C-peptide [26]. Urinary C-peptide levels had a high sensitivity for differentiating MODY
from T1D or T2D [27], but had the disadvantage of being useful only in patients with
adequate renal function.
In our study, significant differences were presented between the C-peptide concentra-
tions of patients with T1D, T2D, MODY, and LADA. Those differences were significant
even after long-term disease duration and despite treatment. In fact, as demonstrated by
Bouche et al., prolonged treatment with insulin increases the C-peptide response [28]. As
mentioned, the majority of C-peptide is metabolized by the kidneys, with 5–10% excreted
unchanged in the urine. In patients with chronic kidney disease, its precise quantifica-
tion is difficult [25]. In our study, despite some differences in creatinine clearance among
groups, all patients had a creatinine clearance higher than 60 mL/min/1.73 m2 . Fur-
thermore, creatinine clearance did not influence C-peptide concentrations in the multiple
regression analysis.
C-peptide concentration declines over time in all types of diabetes [7]. It has been
observed that the prevalence of detectable C-peptide varied from 19% in people diagnosed
before the age of 15 with diabetes duration greater than 15 years to 92% in those with
onset after 35 years and diabetes duration less than 5 years [29]. In China, patients with
LADA had a rapid decline in C-peptide concentrations during the first 5 years of diagnosis,
depending on the GAD autoantibody titration [30]. In T2D, there is a hyperinsulinemic
phase due to insulin resistance that develops before the decline of ß-cells. During the
prediabetes stage, a high proinsulin/C-peptide ratio is observed. After diabetes onset,
C-peptide declines; however, it persists detectably for more than 20 years despite the use of
insulin [29].
In Korea, in 223 patients with diabetes, levels of C-peptide 0.6 ng/mL excluded a
diagnosis of T2D, while C-peptide levels > 3.0 ng/mL made a diagnosis of T1D improba-
ble [31]. Likewise, Katz et al., in 175 patients, identified fasting concentrations of C-peptide
of 0.85 ng/mL (83% sensitivity, 89% specificity) to distinguish the pediatric population with
T1D from the population with T2D [32]. In our study, the cut-off of 0.95 ng/mL achieved
a similar sensitivity (82%), while specificity was lower (77%). Genes et al. evaluated
104 patients with diabetes aged older than 16 years: 24 with HNF4A-MODY, 40 with T1D,
and 40 with T2D; their mean age was 32 years, with fasting glucose levels > 200 mg/dL
and HbA1c > 10%. In this group, C-peptide levels were different between T1D and T2D
(0.86 [0.01–4.61] ng/mL vs. 2.38 [1.05–11.8] ng/mL, p < 0.001) and between T1D and MODY
(with a C-peptide concentration of 1.78 [0.47–5.05] ng/mL, p = 0.003) [33]. Although they
did not perform ROC curves, it is noteworthy that their C-peptide levels are similar to
our proposed cut-off points. In another cohort study carried out in Sweden that included
Life 2024, 14, 550 9 of 13

2734 children recently diagnosed with diabetes, patients classified as T2D had the highest
C-peptide concentration (5.5 ng/mL), followed by patients with MODY (3 ng/mL) and
T1D (0.84 ng/mL) [8]. Despite that our population is quite different from our adult cohort,
with more than 10 years since the diagnosis of diabetes, it supports that lower C-peptide
levels are useful for detecting T1D.
Fasting C-peptide levels in patients with LADA are lower when compared with those
with T2D. However, ß-cell reserves differ among patients with LADA due to heterogeneity
in the severity of the autoimmune process. This has been considered by an international
expert panel that recommends a personalized treatment depending on three categories
of random C-peptide: if levels are less than 0.90 ng/mL (0.3 nmol/L), a multiple-insulin
regimen is suggested; those with levels between 0.90 and 2.0 ng/mL (0.3–0.7 nmol/L)
could be treated with insulin in combination with other therapies in a flexible scheme;
and those with levels higher than 2.0 ng/mL (0.7 nmol/L) could be treated according to
T2D guidelines. In this group, they recommend repeating the C-peptide measurement
if glycemic control deteriorates [34]. In our study, the mean C-peptide concentration
in patients with LADA was 1.87 ng/mL, which suggests that they could benefit from a
combined treatment with insulin and oral anti-diabetic drugs. Most importantly, we defined
a C-peptide cut-off point of 0.82 ng/mL, which allows us to differentiate between T1D and
LADA. Future studies could explore if this cut-off point could differentiate between those
with autoimmune diabetes in longitudinal studies with a larger sample size that includes
other ethnicities.
Thunander et al. evaluated the utility of C-peptides to classify diabetes. They found
that C-peptide was a better discriminator in comparison with age and BMI to identify
those patients with at least one positive autoantibody (anti-GAD and/or anti-IA2). In fact,
upon creating ROC curves (taking as an evaluation parameter the adequate classification
of the patient), they observed that the highest values in the AUC were obtained by C-
peptide, followed by age and BMI (AUC of 0.78, 0.68, and 0.66, respectively) [24]. In our
study, the ROC curve for C-peptide had a greater AUC (0.88), which translates to greater
discriminatory ability in comparison with that previously reported. However, BMI and
age did not have adequate discriminatory ability. The ADA and the European Association
for the Study of Diabetes (EASD) consensus report on the management of T1D in adults
recommend evaluating C-peptide after 3 years since diagnosis in autoantibody-negative
patients. They proposed that a cut-off point of 0.60 ng/mL (0.20 nmol/L) suggests a
diagnosis of T1D, while values higher than 1.80 ng/mL (0.60 nmol/L) indicate T2D [35].
According to our results, a higher cut-off point could be proposed to differentiate T1D from
T2D (0.95 ng/mL), while the cut-off point of 1.65 ng/mL also allows the differentiation
between T2D and MODY in the Mexican population. Being able to adequately differentiate
the type of diabetes that a patient has allows us to provide an appropriate and more
effective treatment. For example, mainly insulin in patients properly diagnosed as having
T1D, sulfonylureas in patients with MODY, or a multi-treatment approach in patients
with T2D.
C-peptide has been correlated with various components of metabolic syndrome. Ha-
ban et al., in a study of older patients with T2D, found significant correlations between C-
peptide and triglycerides, HDL-c, and various relations with different kinds of atherogenic
indices (total cholesterol /HDL-c and TG/HDL-c), as well as BMI and leptin concentra-
tions [36]. In our study, we observed a moderate correlation with fasting glucose both in the
general population and in patients with T2D, showing no significance in patients with T1D
or MODY. C-peptide and BMI showed a slight correlation in T1D and T2D and a negative
correlation with HDL-c in patients with T2D. Considering the above, serum determination
of C-peptide might constitute an efficient tool in the prediction of cardiovascular diseases
in patients with T2D, permitting primary measures of prevention and opportune treatment.
Regarding the determination of autoantibodies in patients with T1D, it has been
reported that the prevalence of anti-GAD oscillates between 60 and 85%, while the deter-
mination of anti-IA2 varies between 70 and 90% of patients [37]. In our population, the
Life 2024, 14, 550 10 of 13

prevalence of anti-GAD was 55% and of anti-IA2 was 42%. The prevalence of anti-IA2 has
not been reported previously in our country. In T1D, there is usually a high percentage
of positivity for anti-GAD at diagnosis, but anti-IA2 positivity rises with disease progres-
sion [38]. However, another study from the same trial found that levels of C-peptide
dropped over time, at least during the first two years [39]. In contrast, in LADA, levels of
both anti-GAD and anti-IA2 decrease between one and ten years of disease duration [40].
In the case of LADA, evidence suggests an inverse relationship between anti-GAD and
C-peptide [41].
The diabetes autoantibody standardization program (DASP) has proposed different
cut-off points, as well as standardization of the reference ranges for the diagnosis of
T1D [42]. In the DASP 2000 workshop, both anti-GAD and anti-IA2 showed high sensitivity
(80 and 58%) and specificity (90 and 100%, respectively) [43]. In contrast, in our study, lower
sensitivity was observed with greater diagnostic specificity for anti-GAD and anti-IA2
antibodies. Zinc transporter 8 (ZnT8) autoantibodies have been recognized as one of the
major anti-islet autoantibodies, present in 63% of patients with T1D and in 27% of patients
with LADA. It has been observed that a more complete autoantibody panel could increase
the accuracy of differentiation between autoimmune and non-autoimmune diabetes [44].
In our institution, we were not able to perform anti-ZnT8 autoantibodies. In fact, its use
is not widespread in Mexico. Furthermore, it is necessary to stress that the determination
of antibodies is costly and is only performed in some research units in our institution;
therefore, we propose the determination of C-peptide as a less expensive diagnostic tool
that is easy to perform and that can be used in primary care facilities.
In regard to limitations, one might consider the scant number of patients with MODY
and that all of them were selected from a single center in a specific region of Mexico. Addi-
tionally, our clinic belongs to a tertiary referral center, where we attend a higher number
of patients with T1D, MODY, and LADA and fewer patients with T2D than expected
nationwide. This could limit the generalizability of the findings, as the population might
not be fully representative of all Mexicans. We expect to establish further collaborations
with different clinics around Mexico and Latin America that will allow us to confirm our
results. Furthermore, the cross-sectional design of the study impedes our ability to evaluate
how the C-peptide level changes over time in patients with T2D, LADA, and MODY. We
plan to perform a longitudinal study to evaluate the utility of C-peptide for monitoring
disease progression in these different types of diabetes, and if its concentrations are related
to response to treatment. Another limitation was the impossibility of performing genetic
studies on our entire population. However, McDonald et al. have suggested that the pres-
ence or absence of islet antibodies, especially anti-IA2, is a good indicator of MODY vs T1D
and that the more expensive genetic testing should only be administered if other clinical
characteristics so warrant it [45]. In addition, we obtained a power calculation higher than
90% when comparing C-peptide concentrations among groups. Another limitation of the
study is the time since the diagnosis of all types of diabetes in the population (10 years).
Previous studies in patients with T1D have reported residual insulin secretion at the time
of diagnosis and within 1–2 years after diagnosis [46]. Likewise, a large cohort from the
T1D Exchange Clinic Network reported that the odds of having detectable C-peptide were
7% lower for every year increase in diabetes duration. Nevertheless, they also observed
that 35% of the participants with 10–19 years since diagnosis still had detectable residual
C-peptide [46]. Haupt et al., in the KID Study, observed that among patients with type
2 diabetes, C-peptide decreased with disease duration of 15–20 years, but still was in the
high normal range even after this time [47]. Chaillous et al. also observed that among
patients with LADA with tight metabolic control, C-peptide concentrations decreased,
irrespective of age, gender, BMI, antibody titers, HbA1c, or treatment modality [48]. At this
point, we must consider that patients in our study had variable ranges of glycemic control.
It is well known that high blood glucose levels stimulate insulin secretion. Despite this,
some studies have observed that chronic hyperglycemia (assessed through HbA1c) does
not influence C-peptide levels [49]. Furthermore, neither diabetes duration nor glycemic
Life 2024, 14, 550 11 of 13

control were associated with C-peptide levels in the logistic regression analysis performed
in our study.
Finally, we must consider that this study was conducted on the Mexican population.
More studies need to be conducted on populations of different ethnicities to corroborate
our findings.

5. Conclusions
C-peptide could be useful for the diagnostic differentiation of patients with T1D, T2D,
and LADA. According to our results, a cut-off point of 0.95 ng/mL could differentiate
between T1D and T2D, while a cut-off of 0.82 ng/mL could differentiate between T1D
and LADA. Despite the significance, the cut-off of 1.65 ng/mL observed between T2D
and MODY remains with a 25% misclassification and requires further investigation. These
results are only applicable to patients with long-standing diabetes.

Author Contributions: Conceptualization, R.A.-C., M.E.N.-G., A.F.-H. and R.A.G.-D.; methodology,

R.A.G.-D., R.M.-G. and N.W.; validation, R.M.-G.; formal analysis, A.F.-H. and R.A.G.-D.; investiga-
tion, R.A.-C., M.E.N.-G. and R.M.-G.; resources, A.F.-H.; data curation, A.F.-H., R.A.G.-D. and N.W.;
writing—original draft preparation, R.A.-C. and M.E.N.-G.; writing—review and editing, R.A.G.-D.
and A.F.-H.; supervision, M.E.N.-G. and A.F.-H.; project administration, A.F.-H. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: This study was conducted in accordance with the Declaration
of Helsinki and approved by the Local Research and Ethics Committee (Comité Local de Investigación
y Ética en Investigación en Salud) of the Instituto Mexicano del Seguro Social (IMSS Registry number
R-2016-3601-9). The procedures are in compliance with local ethical regulations, the Mexican Law of
General Health regarding Health Research, the institutional regulations, and the currently approved
international codes and norms regarding Good Clinical Practice in Clinical Investigation.
Informed Consent Statement: Informed consent was obtained from all subjects involved in this study.
Data Availability Statement: The data analyzed in this study are available from the corresponding
author on reasonable request.
Acknowledgments: The authors thank Susan Drier-Jonas for her assistance with the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.

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