Effect of Protein Supplements on Renal Function and Lipid Profile

Objective: To assess the knowledge, attitude and practice of Saudi population toward the protein supplements, examine the relationships of practice with education level, effect of protein on kidneys and lipid profile, assess the types and source of protein supplements, assess the correct use of protein supplements according to exercise intensity and determine protein content and heavy metals in protein supplements.


Introduction
Nutritional supplements are defined by United States law as products taken by oral to complete total daily requirements.It is increasing in use to 88% globally [1,2].
Protein supplements are one of the most frequently used dietary supplements used by athletes and non-athletes [3,4].Recently whey protein and hydrolyzed collagen have seen heavy marketing.Many people are making them a part of their diet and exercise routines [5].
Collagen is a main structural protein, which is extracted from collagen-rich materials with hot water as gelatin.The common materials used for extracting gelatin include bovine, bones and fish.The further enzymatic hydrolysis of gelatin results in hydrolyzed collagen [6].Hydrolyzed collagen contains all amino acids necessary to retain the normal structure and strength of the skin, connective tissues, tendons, cartilage, bones and joints [7,8].
Milk is made up of two types of protein: casein and whey.Whey is the liquid portion of milk that can be extracted and separated from casein or it can be formed as a by-product when cheese is being made [9].Whey is high use because of its ability to increase muscle mass and reduce body fat [3,4,10].It is extracted, purified and separated to obtain several concentrations that also vary in fat and carbohydrate.Three main forms of whey protein are: concentrate, isolate and hydrolyzed whey protein.Concentrate whey protein contains about (89%) protein and (10%) fat.Filtering this (89%) protein removes more carbohydrate, lactose and fat, and this creates isolate whey protein which has protein at more than (90%) and less fat (0.5%).Hydrolyzed whey protein is fraction to small peptides variable in protein and fat content [5,9].
There is an expectation by consumers the protein supplements that they will meet their dietary and health needs by ingestion of these supplements.There is a belief that the stated amounts of the ingredients contained in them are pure and do not contain any foreign ingredients other than the ones listed on the label of the containers.However, research has proven that, these supplements can be contaminated with impurities.There is an evidence of poor quality control in the manufacture and preservation of these supplements [5].
After tests and research on some of commercially-available protein supplements products, they failed to pass the quality assurance test [11].It was found that, protein powders and drinks contaminated with heavy metals(lead, arsenic, cadmium and mercury).The levels of the contamination were too high.Such a concentration is enough to pose great risk to consumers (5).It affects health of many organs like liver, kidneys, brain, heart and cause blood lipid profile disturbance [12].The users should be informed regarding safety associated with Protein supplements and consider the quality of protein contained in the protein supplements and the possibility of adulteration [11].
Recently, they are increasing in interest and use in Saudi Arabia [13,14].Approximately 68.4% of 316 participants had used protein supplements, and did not believe that these agents cause harm [15].Additionally, Jawadi et al., (2017) study included 299 participants, who consume a total of 25 supplements, showed that the most common used supplements is whey protein (22.1%) [14].
The Recommended Daily Allowance (RDA) of protein requirement forgeneral people is 0.8 g/kg/ day.This amount is adequate enough for general fitness or light exercise intensity, but the amount can still be raised to 1 g/kg/day [3,16].Increasing requirements with increase exercise intensity, for moderate exercise intensity 1.4 g/kg/day and high volume of intensity 1.7g/kg/day according to the international society of sports nutrition (ISSN) [17].The recommended daily amount of hydrolyzed collagen supplements is 10 grams [18].
It is recommended that the protein requirements should be reached through diet only [17].On the other hand, if they intake protein supplement higher than recommended amount, they will be more susceptible to its side effects [19,20].
High protein consumption may be deleterious on kidneys health [19].The kidneys play a central role in balancing the effects of protein metabolism [21].The increase in urea, albumin and calcium and oxalate excretion leads to hyper-filtration, which renders the kidneys more prone to chronic kidney disease (CKD) in healthy individuals and exacerbates issues in patients already suffering from kidney dysfunction [22,23].
Most of these protein supplements contain creatine.creatine can develop interstitial nephritis which leads to renal failure.These side effects can be developed in a rather short period and even when taken as a small dosage [23,24].
The increase in protein consumption is one of causes ofkidney stone disease and it's a risk factor of chronic kidney disease.This can be related to increase in acid excretion and lower renal PH; which is caused by calcium, oxalate and urea excretion [21,26].
Hydroxyproline is a non-essential amino acids (NEAA) mainly contained in collagen [27].It gives strength to the protein collagen in bones, cartilage, tendons and skin, but there is a major risk factor involved in its consumption.It can lead to calcium oxalate stone formation which can end in renal failure [23,26].
High intake of fruits and vegetables greatly reduces the risk of stone formation because of their high content of potassium and magnesium that helps in reduction urinary calcium excretion [21].Constant exercise and high water intake at least two liters a day can protect kidneys and slow renal decline [28,29].
As well, high protein consumption has a negative effect on blood lipid profile.It elevates low density lipoprotein (LDL) and triglyceride (TG) increasing risk of cardiovascular complications [30].Intense and constant exercise can decrease this disturbance [31].
Therefore, there is a direct relationship between abuse of dietary protein supplements and the disturbance in renal function and lipid profile.
The aim of this study was to assess the knowledge, attitude and practice of Saudi population toward the protein supplements, to examine the relationships of practice with education level, effect of protein on renal function and lipid profile, to assess the types and source of protein supplements, to assess the correct use of protein supplements according to exercise intensity and to determine protein content and heavy metals in protein supplements.

Materials and Method
The study has two phases; including survey submitted to protein supplements users and determines protein content and heavy metals in protein supplements products collected from shops and purchased from Internet.The user's survey will investigate three parts: demographic data, usage protein supplements and perception of risk.Samples of the products sent to the laboratory for quantitative analyses.

Inclusion criteria
Saudi Arabian participants who are more than 13 years old and protein supplements users for at least 3 months were included in this study.

Exclusion criteria
Participants, who used protein supplements less than 3 months, are less than 13 years old or smokers and participants who has hypertension, renal, heart diseases before administration of protein supplements were excluded from this study.

Study design
A national cross-sectional, standardized and confidential survey was conducted on Saudi Arabian users of protein supplements during October-December, 2017 in Qassin region.

Questionnaire
The questionnaire designed in English and later translated to Arabic by a professional translator and reviewed for consistency by the investigators.The questionnaire includes the demographics of protein supplements users, the source of getting protein supplements, the side effects related to protein supplements used and perception of the users to the associated risk.The questionnaire pre-test for validity, reliability and checked if it is easy for the interviewers to understand the instructions and flow of questions.Prior to pre-testing in the field, the interviewers undergo trainings and they provide with instructions about the survey.

Measured correct quantity of protein supplements
According to daily protein requirements for physical activity; general people without doing any exercise(0.8g/kg/day) general fitness (1 g/kg/day), moderate amount of intense training (1.4 g/kg/day) and high volume of intense training (1.7 g/kg/day) [11,16,17].The recommendation for hydrolyzed collagen is 10g per day [16].

Chemical analysis
Products collected from shops and online retailers were analyzed to determined total protein and heavy metals content.Determination of heavy metals content in protein supplements by inductively coupled plasma mass spectrometry (ICP-MS) and for total protein content using kjeldahl Method according to association of official analytical chemists AOAC methods (991.20) for whey protein samples and (981.10) for hydrolyzed collagen sample [32].

Statistical analysis
Data was analyzed using the (SPSS) version 24.Ascertain the significance of differences between mean values of two continuous variables was analyzed by t-test.Chi-Square test was used to determine associations between different variables.P-value ≤0.05 will be considered significant.
The average weight of all participants was 70.92 kg, and the average length of all participants was 164.62 cm.
Most of the participants had a bachelor's degree of (76.9%) and (17.3%) had Intermediate or high school while (5.8%) had postgraduate studies.

Types of protein supplements
Table 2 shows that more than half of the participants (51.8%) were taking isolated whey protein, while just over a quarter of the participants with (27.93%) were taking concentrated whey protein, (11.3%) of the participants were taking collagen and (9%) of the participants were taking other protein supplements.

Usage of protein supplements
Table 3 shows that more than half (55%) of the participants used protein supplements for bodybuilding, while (30.9%) were taking protein supplements for cosmetic and (14.1%) were taking protein supplements for other purposes.On the other hand, (46.7%) of participants got protein supplements online, while (33.9%) of participants got it through markets and (19.3%) got protein supplements through pharmacies.

Knowledge about the risk related to protein supplements
Table 4 shows that one hundred and fourteen (28.6%) of our participants knew that the exceeding dose of protein supplements increase the side effect.Two hundred and thirty-two (58.3%) of our participants knew that the protein supplements affect kidney.One hundred and seventeen (29.4%) knew that the protein supplements affect lipid profile.Three hundred and nineteen (80.2%) of our participantsknew that frequent drinking water may reduce the side effect.Two hundred and sixty-five (66.6%) of our participants knew that exercise intensity may reduce the side effect.

Attitude towards protein supplements use
Figure 1 shows about (86%) of participants took protein supplements without any advice from doctor or pharmacist, while about (14%) of the participants took protein supplements with taking advice.Practice of protein supplements users Table 5 shows (43%) of participants were taking protein supplements daily, (30.7%) were taking them four to six times, while (26.4%) were taking them once to three times.About (54.3%) of parti-cipants the protein correctly, while (45.7%) of the participants took the protein in the wrong way.
Approximately half of the participants (49.7%) of participants took the protein supplements for (≥ 3-6 months), while (14.3%) of them took it for (more than 6 months), (11.6%) took the protein supplements for one year and (24.4%) of participants took the protein supplements for more than a year.About (28.9%) of the participants took 1-4 cups of water per day, (33.1%) took 4-6 cups per day (23.4%)took 6-8 cupsper day and (14.6%) of the participants took more than 8 cups per day.

Relationship between practice and education level Table 6
demonstrates that, There is no significant relationship between education level of our participants and the frequent usage of protein supplement per week (x 2 = 4.651, P= 0.325).
Also, there is no significant relationship between education level of our participants and the method of protein consumption (x 2 = 1.816,P = 0.403).
There is no significant relationship between Education level of our participants and the duration of protein consumption (x 2 = 7.473, P = 0.279).
There is no significant relationship between Education level of our participants and the amount of water per day (x 2 = 11.088,P= 0.086).

Relationship between exercise intensity and the method of protein consumption
Table 7 shows that there is a significant relationship between exercise intensity and the method of protein consumption (x 2 = 14.944,P-value = 0.001) which means that the protein consumption method varies according to exercise intensity and therefore affects the method of protein consumption.
Table 8 shows that only eighty five (21.4%) of our participants found change in RFT, while Twentyeight (7.0%) of the participants found no change in RFT after using the protein supplements.Relationship between practice and the change in RFT Table 9 shows that there is no significant relationship between type of protein supplements and the change in RFT(x 2 = 2.775, P= 0.428).There is a significant relationship between usage of protein per week and the change in RFT (x 2 = 8.936, P= 0.011).
There is a significant relationship between the appropriate use of protein consumption and the change in RFT (x 2 = 10.394,P= 0.002).
There is no significant relationship between the duration of protein consumption and the change in RFT (x 2 = 5.331, P= 0.149).
There is a significant relationship between Exercise intensity and the change in RFT (x 2 = 11.795,P= 0.008).
There is a significant relationship between the amount of water per day and the change in RFT (x 2 = 31.756,P= 0.00), which mean that increase the consumption of water improve renal function.

Type of lipid profile changes after using the protein supplements
Table 10 demonstrates that only thirty-three (8.3%) of our participants found change in lipid profile after they were taken the protein supplements, while thirty-eight (9.5%) found no change lipid profile after using the protein supplements.
Only fifteen (3.8%) of our participants found that they had an increase in total cholesterol, while eight (2.0%) of the participants found that they had a decrease in total cholesterol after using the protein supplements.
Only nine (2.3%) of our participants found that they had an increase in triglycerides, while six (1.5%) of the participants found that they had a decrease in triglycerides after using the protein supplements.
Only twelve (3.0%) of our participants found that they had an increase in low-density lipoprotein, while eleven (2.8%) of the participants found that they had a decrease in low-density lipoprotein after using the protein supplements.
Only five (1.3%) of our participants found that they had an increase in high-density lipoprotein, while nine (2.3%) of the participants found that they had a decrease in high-density lipoprotein after using the protein supplements.There is a significant relationship between the usage of protein per week and the change inlipid profile (x 2 = 6.543,P= 0.038).

Relationship between practice and the change in lipid profile
There is a significant relationship between the method of protein consumption and the change inlipid profile (x 2 = 3.895, P= 0.048).
There is no significant relationship between the duration of protein consumption and the change inlipid profile (x 2 = 2.107, P= 0.550).
There is no significant relationship between Exercise intensity and the change inlipid profile (x 2 = 5.479, P= 0.140).
There is a significant relationship between the amount of water consumed per day and the change inlipid profile (x 2 = 14.193,P= 0.003) that means increase of water consumption per day improve lipid profile.

Relationship between practice and the type of change in lipid profile
Table 12 demonstrates that there is no significant relationship between practice of protein supplements and the type of change in lipid profile.

Discussion
Protein supplements are one of the most frequently used dietary supplements used by athletes and nonathletes for performance enhancement and tissue repair [3,4].It is divided into two sources plant protein (soy, bean and rice) or animal protein (milk, eggs and bovine colostrums) [9].Protein supplements contain essential amino acids (EAA) like (lysine, leucine and methionine) and non-essential amino acids (NEAA) (glycine, proline, and alanine), hydroxy-proline is NEAA derivative formed through post translational protein modification during hydroxylation of proline [8].Quality of protein sources is diverse according sential amino acids profile.Animal protein contains high amount of essential amino acids compared to plant protein, so it's classified as high quality protein.Additionally the quality of protein supplements products varies depending on the manufacturing process [9,33].
In this study, the demographic characteristics of participants showed more than half of the participants (56.3%) were in the age (21)(22)(23)(24)(25)(26)(27)(28)(29)(30), while (32.9%) of the participants were in the age (31-50) and (10.3%) of the participants in the age (18)(19)(20) years.Previous studies found that most of participants were adolescent [34,35,36].Also, in the present study we found that two-thirds of the participants females (60.1%), while (39.9%) of males participated.El Khoury and Antoine-Jonville (2012) found that the male users of protein supplements were more than females because they use it for muscle building and performance enhancement.
Our study showed that, most of the participants had a bachelor's degree of (76.9%) and (17.3%) had Intermediate or high school while (5.8%) had postgraduate studies [37].Also, most of the participants (32.2%) were in general fitness, twenty eight (28.9%) did not perform exercise, twenty four (24.9%) perform moderate exercise while fourteen (14.1%) in perform strength exercise.More than half (55%) of the participants used protein supplements for muscle building, while (30.9%) were taking protein supplements for cosmetic and (14.1%) were taking protein supplements for other purposes.In contrast, El Khoury and Antoine-Jonville (2012) found that most of participants use protein supplements for muscle building in high volume exercise intensity.These observations originate from misconceptions concerning effectiveness of protein supplement in muscle building without exercise or in general fitness [37].
In the current study, (46.7%) of participants got protein supplements online, while (33.9%) of participants got it through markets and (19.3%) got protein supplements through pharmacies.The online purchase of protein supplements in this proportion of users may be exposed to the risk of using unregulated products with no formal test of their quality, especially from substandard retailers.And they increased education and awareness among protein supplements users as this poses a significant danger to their health is required [36].
We found that two hundred and forty-eight (71.4%) of our participants did not know that the exceeding dose of protein supplements increase these side effect.Two hundred and thirty-two (58.3%) of our participants knew that the protein supplements affect kidney.One hundred and seventeen (29.4%) knew that the protein supplements affect lipid profile.In agreement with these findings El Khoury and Antoine-Jonville (2012) found that a majority of protein supplements users were unaware of the negative health implications when used in excess of the recommended dose.The high rate of consumption may be reflective or indicative of a pattern of dangerous and inappropriate use.Lack of adequate and proper knowledge explains the lack of awareness among users [37].
In the present study, majority of our participants (86%) took protein supplements without advice doctor or pharmacist, while about (14%) of the participants took protein supplements with taking advice.In agreements with our results, Jawadi et al., (2017) found that, users obtained their information regarding the use of supplements from coaches, online websites, journals and other sources which are mostly unreliable.Only 13.3% of participants took a physician's advice [14].In contrast, Saudi study finding that reliable sources of information obtained from physicians (45.9%) [38].
Our study showed that, the participants who consumed protein supplements in exceeding doseor in long durationaffect renal function especially for those who did not make exerciseor did not drink adequate amount of water.In agreement with So et al., (2016), who found that participants who consumed protein more and without physical activity were more likely to renal diseases [29].High protein consumption causes dehydration which affects renal health [39].Sontrop et al., (2013) found that, daily water intake more than two liters has protective effect on kidney [28].
Also, high protein consumption with light exercise intensity affects blood lipid profile but this effect is not clear and may be due to the lower number of participants who performed routine test on the lipid profile.Amini et al., (2016) found that, the disturbance in lipid profile due to fat contained in protein supplements products [30].High exercise intensity is preventing this effect on lipid profile [19].
The percentage of protein content in isolated whey protein is (68.18%),concentrated whey protein contain (72%) and hydrolyzed collagen containing (100%) protein.By Kjeldhl analysis, we found that isolated whey protein contain (80.55%) of protein while concentrated whey protein contain (77.2%) of protein hydrolyzed collagen containing (91.25%).So, the percentage of protein content found by Kjeldhl analysis more than the percentage of protein content which labeled on containers.kjeldhl analysis showed that hydrolyzed collagen sample had a less protein percentage than container label.These is increase the potential risk of these products on renal function due to these effects on glomerular filtration rate and kidney stone formationrelated to increase in acid excretion and lower renal PH; which is caused by calcium, oxalate and urea excretion it's a risk factor of chronic kidney disease [19,21,26,39].On other hand, some of supplements manufacturer reduce the amount of expensive components and added other components less cost [40].
The present study found that, in 100 grams of isolated whey protein contains arsenic (83 μg/kg), cadmium (8 μg/kg), mercury (<10 μg/kg) and lead (18 μg/kg), while hydrolyzed collagen sample contains arsenic (101 μg/kg), cadmium (<2 μg/kg), mercury (<10 μg/kg) and lead (49 μg/kg).Isolated whey protein sample contaminated with cadmium while hydrolyzed collagen sample contaminated with arsenic and lead.Heavy metals are highly toxic that they may have cumulative harmful effects especially to the kidneys and heart.The mechanism of heavy metals toxicity still unknown, although inhibition of enzymes, impaired antioxidants and make imbalance in redox system [12].Arsenic, mercury and cadmium exposure affects the kidney by impaired proximal tubular cell function and glomerular damage [12,41].Also, they cause adversely effect on blood lipid profile.Increased level of serum TG, TC, LDL and reduced HDL that is leads to atherosclerosis [41,42].Lead exposure affects the mechanical and electrical cardiac activity and raise arterial pressure that develop cardiovascular disease [12].

Conclusions
The current study reveals an evidencefor lacking of the awareness, negative attitude and abuseof protein supplements, demonstrates necessity to further education regarding the use and purchasing of protein supplements to ensure the quality of these products.Additionally, abuse of protein supplements has an adverse effect on renal function and lipid profile.
The percentage of protein content found by Kjeldhl analysis is more than the percentage of protein content which labeled on containers and the samples contaminated with heavy metals which means that there are adulteration in the industry of protein supplements products, that is increased the harmful effects of the protein supplements on the renal function and blood lipid profile.

Table 4 .
Knowledge about the risk related to protein supplements (n=398).

Table 8 .
Percentage of participants who found a change in RFT after using the protein supplements (n=113).

Table 6 .
Relationship between practice and education level (n=398).

Table 7 .
Relationship between exercise intensityand the method of protein consumption (n=398).

Table 9 .
Relationship between practice and the change in RFT (n=113).

Table 10 .
Type of lipid profile changes after using the protein supplements (n=71).

Table 11
demonstrates that there is no significant relationship between type of protein supplements and the change in lipid profile (x 2 = 4.408, P= 0.221).

Table 11 .
Relationship between practice and the change in lipid profile(n=71).

Table 13 .
Percentage of protein find in protein supplementssamples using kjeldhl method.

Table 14 .
Heavy metals determination in protein supplements samples using (ICP-MS).