Omega 3 Long Chain Polyunsaturated Fatty Acids, Enriched Mediterranean Diet and Vitamin D Supplementation in Childhood Type 1 Diabetes: One Year Case-cohort Study
Phase: PHASE2
Conditions: Type 1 Diabetes Mellitus
Sponsor: Azienda Ospedaliero Universitaria Maggiore della Carita

01/01/2017 
 
Vitamin D and fatty acids Ω3: role in the "honeymoon" of the Type 1 Diabetes of the child 
 
The weight of diabetes on the child, on the family and in society 
 
Type 1 diabetes (T1D) is an autoimmune disease in which there is a progressive reduction of insulin- 
secreting β-cells that involves the need to undertake replacement therapy with exogenous insulin. 
 
The therapy consists of the administration of insulin in multiple administrations per day by pen or 
continuous infusion under the pump and requires frequent monitoring of capillary blood glucose or 
continuous glycemic monitoring with medical devices. T1D is burdened by important long-term 
complications: neurological, ocular, renal microangiopathic, which make diabetes the leading cause of 
chronic renal failure and dialysis, and acquired blindness. In addition, the pathology determines diabetic 
macroangiopathy, which leads to an increased risk of heart attack, macrovascular damage especially in the 
lower limbs with consequent diabetic foot and atherosclerotic complications of specific cardiac, cerebral 
and genital districts. These micro and macro-angiopathic complications, together with the continuous 
increase in the incidence and prevalence of diabetic pathology, cause a huge social and individual cost that 
currently represents a significant resource commitment, and will become an unsustainable health care 
expenditure in the years to come. 
 
Data from the Pediatric Regional Register (0-15 years), reported in Literature, provide a precise assessment 
of the incidence of T1D in the Piedmont Region: from these data we can see an increase of 3.3% annually, 
with a consequent increase in direct costs of diabetes (in Italy for the NHS in 2012 direct costs over € 9 
billion, 57% for hospital access, 30% for drugs and devices, 13% for outpatient care) and indirect and 
immaterial, which are even higher, even if hardly quantifiable. There was also a greater increase in 
incidence in pre-school and immigrant children, especially in relation to the dark color of the skin that limits 
the vitamin D biosynthesis. 
 
The previous consideration, together with the prevalence of widespread vitamin D deficiency usually 
detected at the onset of T1D, led to a causal relationship between vitamin D deficiency and the genesis of 
diabetes. The essential role of this vitamin consists in the immunoregulatory activity on T-lymphocytes. 
Today, vitamin D supplementation in every child with diabetes, with 1000 IU / day (corresponding to the 
current indications of the Endocrine Society, IOS and SIPPS), and the monitoring of the actual serum 
concentration [as 25 (OH) D3] in the range 30-50 ng / ml, is a therapeutic act that is consolidating in the 
therapy of T1D. 
 
In addition to a genetic condition of polygenic risk, which finds the greatest risk related to specific 
predisposing HLA alleles, there are no known causes of this autoimmune pathology, ie an event, 
presumably environmental, conditions the increased incidence of the disease especially in fast-moving 
countries socio-economic evolution. By logical deduction, genetic predisposition is stable, and the increase 
in incidence of T1D must be related to environmental factors (pollutants, infections, especially viral, or food 
changes), since the increase in T1D is observed in countries of all over the world, and in particular in 
developing countries. There are many possible environmental causes of the increase of T1D. 
 
A vitamin D and omega 3 (Ω-3) deficiency status is reported in the general population, including the 
pediatric population. The lack of Ω-3 in our power supply could represent an environmental cause or an 
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amplifying factor a trigger event. In any case, the ratio between Ω-6 and Ω-3 has shifted from 1: 1 a few 
decades ago, to a current 10: 1 or 25: 1. In particular, a case-control study of Stene et all. in Norway (2000) 
showed that the intake of cod liver oil by the mother during pregnancy or during the first year of life was 
associated with a reduced risk of developing T1D. Stene concluded that vitamin D and long fatty acids 
omega-3 (Ω-3), eicosapentenoic acid (EPA) and docosahexaenoic acid (DHA) have a protective effect 
against the development of T1D. In a further study, Stene and Joner (2003) found that dietary 
supplementation of cod liver oil during the first year of life was significantly associated with a lower risk of 
developing T1D during childhood (OR 0.7, p <0.001). This is consistent with a protective role played by 
vitamin D and Ω-3 during pregnancy and early childhood. 
 
Our experience in this regard was initially focused on the case of an 8-year-old boy with T1D who, after the 
classic onset of the disease, has progressively reduced his insulin needs. To date, at 24 months from the 
onset, persists a state of excellent metabolic compensation with a minimum and single administration of 
insulin a day equal to 3 U. To this child, after the diagnosis, once achieved a good metabolic compensation, 
were administered Ω-3 and vitamin D. This result is considered to be different from the usual course of the 
disease that entails, a frequent (but not constant) reduction of the insulin requirement (so-called 
"honeymoon period") to less than 0.5 IU / Kg / day, then followed, invariably by a progressive increase in 
insulin necessary for the metabolic compensation. The "honeymoon" phenomenon underlies a resumption 
of the secretive function of the pancreatic tissue residual to the destruction of β-cells mediated by cells 
infiltrating the pancreas insulae, and by a series of tissue cytokines. 
 
Some children with T1DM in the wake of the published anecdotal case, have already spontaneously begun 
to supplement with omega 3. 
 
At the onset of T1D about 10-30% of β-cellular tissue is still active; the usual increase in insulin needs 
following onset, corresponds to the further depletion of this residual quota, due to the progression of the 
inflammatory autoimmune process (insulitis), potentially counterbalanced by administration of 
immunosuppressive drugs. However, immunosuppressive drugs can not be used in the pediatric age, due to 
possible side effects, and even in young adults, the research trials have so far not yielded significant results. 
There are no side effects in the controlled administration of vitamin D, nor in food supplements such as Ω-3 
fatty acids. 
 
Usually the honeymoon phenomenon is exhausted within a year, even if a residual amount of secretion 
persists at least up to two years from the onset. The endogenous insulin secretion is quantifiable as C- 
peptide, which represents the conjugation peptide between chain A and B of pro-insulin, which after free 
cleavage in the portal circle molecules of C-peptide and insulin in molar ratio 1: 2. Unlike insulin that is 
rapidly degraded in the liver and tissues, the C-peptide passes the liver and is filtered by the unchanged 
renal glomeruli in the urine. C-peptide is not present in exogenous insulin of pharmacological origin. The 
endogenous insulin secretion is quantifiable as C-peptide. A persistence of C-peptide (also dosages 0.2 ng / 
ml) reduces the risk of complications, improves the metabolic compensation and reduces the number of 
hypoglycemic episodes, so at least one of its maintenance would be an important objective to preserve. 
 
 
 
 
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The study is preliminary and aimed at identifying whether a therapy with Ω-3 could be of support in T1D 
especially in the first years of illness, when a persistence of a residual β-cell mass is presumable and it is 
possible to act better on the pro-inflammatory component (insulitis). 
 
 
 
 
Purpose of the study 
 
The aim of the study is to evaluate whether a controlled administration of vitamin D and Ω-3, in the early 
years of the onset of T1D, can preserve the residual β-cell mass, evaluable with a reduced insulin need for a 
long time, with a persistent level of C-peptide, a lower glycemic variability compared to children who do 
not carry out this supplementation. 
 
Secondary assessment is to perform a baseline assessment of the AA / EPA ratio in children with type 1 
diabetes. 
 
 
Subjects and Methods 
 
The subjects will be recruited at the Diabetology Clinic of the Pediatric Clinic of the AOU "Maggiore della 
Carità" of Novara. 
 
In particular, two groups of subjects will be included, subject to the signing of the informed consent: 
 
1°,2°,3° the groups, with T1D onset in 2014, 2015, 2016, retrospectively were evaluated as historical 
controls before 2017. 
 
4 group°: all children with T1D with disease onset in the years 2017, will receive omega 3 supplement (EPA 
+ DHA 60 mg / kg / of), and will be compared from a metabolic point of view (in particular for the needs of 
insulin and glycated hemoglobin) according to the scheme outlined with the pre-supplementation data, 
deduced from the medical records retrospectively. 
 
1°,2°,3° groups: alter 2017 were allowed to enter in the study and were prospectively evaluated 
 
supplementation omega-3 
 
↓ 
 
Group 1 
Group 2 
Group 3 
Group 4 
new onset 
vs Group 1 
vs Group 2 
vs Group 3 
vs Group 1 
vs Group 2 
 
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Vitamin D and fatty acids Ω3: role in the "honeymoon" of the Type 1 Diabetes of the child 

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vs Group 1 
2014 
2015 
2016 
2017 
2018 
 
 
The analysis will allow to quantify the effects of omega-3 at different distance from the onset in terms of 
metabolic advantage (insulin units / Kg / die, glycated hemoglobin, etc.) and to evaluate if and how much 
supplementation after the onset may give metabolic advantages compared to other patients not yet 
supplemented. 
 
 
To the T0, T3, T6 and T12 will be detected anthropometric parameters (weight, height, BMI, Tanner Stages, 
PAOS / PAOD), insulin therapy in progress (IU / Kg / die) with MDI (multiple daily injection) or CSII 
(continuous subcutaneus infusion) and metabolic parameters (C-peptide, coagulation, lipid profile, basal 
blood sugar, glycated hemoglobin, vitamin D). The C-peptide in particular allows us to evaluate the residual 
cell function, which provides a "direct" measurement of endogenous insulin secretion. The fasting C- 
peptide / glycemia ratio will also be evaluated, as an estimate of residual insulin secretion. Glycated 
hemoglobin is an expression of blood sugar in the last three months. A value of less than 7.5% is a sign of 
good compensation. 
 
In addition, the glucometer data will be downloaded at each visit to evaluate the glycemic variability 
parameters (mean glycemia, DS). A DS of less than 50% of the mean blood glucose represents an index of 
stability. The use by all patients of the same meter, delivered at the time of inclusion in the study, will 
eliminate the bias given by the diversity of the medical device. Indication will be given to perform pre-meal 
glycemic controls three times a day, and occasionally after 2 hours from the meal, in order to calculate the 
glycemic variability. 
 
 
An initial dosage of the blood levels of AA, EPA and DHA (and relative ratio) will be carried out by capillary 
sampling and application of a few drops of blood on absorbent paper for all subjects. Fatty acids are 
measurable in whole blood, with a lipidogram, in which each component of FFA is selectively differentiated, 
and then quantified the individual fractions. In particular we will evaluate the ratio between 20: 4 Ω-6 
arachidonic acid (AA), precursor of pro-inflammatory cytokines, and EPA, precursor of anti-inflammatory 
cytokines; this ratio represents a balance between the effector pre-metabolites of inflammation, which 
determine the apoptotic degeneration of β-cells, and the anti-inflammatory ones, which preserve a cellular 
share with an insulin secretive function. This value AA / EPA <3 is proposed in Literature for adults or 
patients with other diseases, but has also been used for children. 
 
The measurement will be carried out free of charge at the Department of Pharmacological and 
Biomolecular Sciences (DiSFEB), Faculty of Pharmaceutical Sciences, University of Milan. 
 
 
 
 
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Vitamin D and fatty acids Ω3: role in the "honeymoon" of the Type 1 Diabetes of the child 

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An assessment of the dietary intake of EPA and DHA fatty acids will be carried out for all the subjects 
included in the study through an interview with the dietician and the compilation of a frequency food 
questionnaire. The collection of the food questionnaire will take place at the time of enrollment (T0) and 12 
months after supplementation with omega 3. All healthy control subjects will be submitted to the food 
questionnaire. Nutritional indications will be given to all families in order to optimize an optimal dietary 
introduction of fatty acids as indicated by the LARNs. 
 
 
Supplementation 
 
The intervention under study is only the administration of omega-3, to all patients with onset in recent 
years (2014-2018) with a contribution of 60 mg / kg / day of the sum of EPA and DHA (corresponding 
respectively to acid eicosapentenoico and docaesoenoico). The AA / EPA ratio will be monitored 
(arachidonic acid, Ω-6 / eicosapentenoic acid Ω-3), to reach the optimal range <3, as indicated in the 
literature. 
 
The chosen preparation will be omega-3, of extraction from fish, with a high concentration and purified to 
exclude mercury and lead, commercially available in stores and supermarkets. EnerZona. Equipe Enervit SrL 
will provide free Ener Zone, in capsules, from EPA 400 mg + DHA 200 mg, or capsules from EPA 200 mg + 
DHA 100 mg, or liquid solution, for subjects unable to swallow capsules or intolerant gluten capsules 
themselves; this will allow us to provide precise delivery and the same preparation to each patient. 
 
All of our patients are supplemented with vitamin D. As vitamin D also has an immunomodulatory role it 
will be monitored in the study, and being frequently low at onset will be given at 1000 IU / day, as indicated 
in prophylaxis for children and adolescent; its level [as 25 (OH) D] will be controlled to reach the optimal 
values (established by Endocrine and other scientific societies) of 40-50 ng / ml, and possibly increased or 
reduced. 
 
Inclusion criteria: 
 
All children with autoimmune T1D, debuted in the years selected at the Pediatric Clinic of Novara. Some 
children with T1D in the wake of the published anecdotal case, have already spontaneously begun to 
supplement with omega 3, and will be excluded from the study. We have primarily considered our series of 
children with T1D onset according to the ADA, ISPAD and SIEDP criteria of the years 2015, 2016 and 2017, 
since insulin secretive activity persists for at least two years from the onset. 
 
Exclusion criteria: 
 
pathologies such as: 
 
• coagulation defects 
 
• renal diseases (nephrotic syndrome, calculosis, renal cysts, etc.) 
 
• sarcoidosis, histoplasmosis 
 
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• endocrinological diseases (hyperparathyroidism) 
 
• tumoral pathologies or tuberculosis 
 
Gluten intolerance (CD) or thyroiditis in T1D are not exclusion criteria. 
 
 
 
Chronology of investigations and time of execution of the study 
 
T.0 Signature of informed consent. Baseline auxological and metabolic evaluation (IU / Kg / day exogenous 
insulin) by means of examination, assessment of dietary intake through food questionnaire and interviews 
with dietician, blood tests (vitamin D, AA / EPA, C-peptide, basal glycemia, glycated hemoglobin, lipid 
profile, coagulation). Discharge of glucometer data for glycemic variability. Start supplementation with 
vitamin D, in case of deficiency, if subject not already supplemented. Begin supplementation with EPA + 
DHA, in case of deficiency, if willingness to recruit. 
 
T.3 at 3 months from the beginning of the supplementation, only for the subjects supplemented with 
omega 3. Auxological and metabolic evaluation (IU / Kg / day exogenous insulin) by examination, blood 
tests (AA / EPA, C-peptide, basal glycemia, glycated hemoglobin, lipid profile, coagulation). Discharge of 
glucometer data for glycemic variability. 
 
T.6 to 6 months from start of supplementation, only for subjects supplemented with omega 3. Auxologic 
and metabolic evaluation (IU / kg / exogenous insulin) by examination, blood tests (AA / EPA, C-peptide, 
basal blood sugar, glycated hemoglobin, lipid profile, coagulation). Discharge of glucometer data for 
glycemic variability. 
 
T.12 At 12 months from the beginning of the study, will be re-evaluated for all subjects, all the previous 
auxological, metabolic and biochemical parameters and a new evaluation of the dietetic dike of fatty acids 
will be carried out. 
 
 
Study security and caution of the subject included 
 
The safety of the therapy with supplements is reflected in the extensive experience of using cod liver oil 
which in the past has been a routine supplement for children for several years. 
 
High doses of Ω-3 over 5 g / day could result in a longer bleeding time, which is why coagulation monitoring 
is required, even if supplementation is required at doses of less than 5 g / day. With the use of Ω-3 a 
reduction of triglyceride levels is observed, which could represent a clinical advantage for the subject and a 
precise therapeutic objective often sought by the practitioners. A triglyceride evaluation will be performed 
pre-supplementation and at 12 months from the beginning of the same. 
 
 
Financial resources and expected costs 
 
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Vitamin D and fatty acids Ω3: role in the "honeymoon" of the Type 1 Diabetes of the child 

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Enervit supplies Ener Zone Omega 3 RX in capsules containing each EPA 400 mg + DHA 200 mg and in liquid 
formulation for younger children and / or with concomitant celiac disease. Ascensia Diabetes Care supplies 
Conturnext USB and Conturnext strips, to allow frequent blood glucose measurements, with accuracy and 
precision. 
 
The blood chemistry tests are part of the normal T1D treatment routine. The dosage of the different 
metabolic and hormonal parameters at the time T1 will be borne by the Pediatric Clinic. The AA / EPA 
report will be determined by the Department of Pharmacological and Biomolecular Sciences DiSFEB, 
Faculty of Pharmaceutical Sciences of the University of Milan free of charge. Other possible findings as 
coagulation will be calculated as expenditure "study use" of the Pediatric Clinic. 
 
 
DATA PROTECTION 
 
to. Blood samples collected on Guthrie cardboard sent to Milan will be collected with the informed consent 
of the parents. 
 
b. For the protection of the subject and clinical data, withdrawals will be identified with a code. 
 
c. The clinical data will be managed by clinical referents inside Pediatrics (Dr. F. Cadario, Dr. S. Savastio) and 
the name of the subject will be identified by initial name and surname 
 
d. The withdrawals will be stored in the Laboratory of Milan Pharmacy University of the Maggiore Hospital 
under the exclusive control of the laboratory referents (Dr. Ssa Rizzo A.M.) 
 
 
Statistic analysis 
 
The data will be expressed as a mean ± SD. For continuous variables, the variations between the groups are 
compared by means of the averages calculated with the non parametric Wilcoxon and Mann-Whitney U 
tests. The variability of the biochemical parameters over time will be evaluated by means of tests for 
repeated measurements. A correlation analysis will be performed with the Pearson correlation test, after 
adequate logarithmic transformation of the parameters when necessary. Multiple regression analysis will 
be used to evaluate associations between variables. Statistical significance will be assumed for values of p 
<0.05. All statistical analyzes will be performed with the SPSS program for Windows version 22.0 (SPSS INC; 
Chicago, IL, USA). 
 
 
 
 
 
 
 
 
 
 
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Vitamin D and fatty acids Ω3: role in the "honeymoon" of the Type 1 Diabetes of the child